Hemagglutinin polypeptides, and reagents and methods relating thereto

ABSTRACT

The present invention provides a system for analyzing interactions between glycans and interaction partners that bind to them. The present invention also provides HA polypeptides that bind to umbrella-topology glycans, and reagents and methods relating thereto.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.13/253,060 filed Oct. 4, 2011, which claims the benefit of U.S.Provisional Application No. 61/389,639 filed Oct. 4, 2010, each of whichare incorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under grant numbersGM57073 and U54 GM62116 awarded by National Institutes of Health. TheUnited States government has certain rights in this invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 21, 2017, isnamed 0492611-1326(MIT14274)_SL.txt and is 915,643 bytes in size.

BACKGROUND

Influenza has a long history of pandemics, epidemics, resurgences andoutbreaks. Avian influenza, including the H5N1 strain, is a highlycontagious and potentially fatal pathogen, but it currently has only alimited ability to infect humans. However, avian flu viruses havehistorically observed to accumulate mutations that alter its hostspecificity and allow it to readily infect humans. In fact, two of themajor flu pandemics of the last century originated from avian fluviruses that changed their genetic makeup to allow for human infection.

There is a significant concern that the current H5N1, H7N7, H9N2 andH2N2 avian influenza strains might accumulate mutations that alter theirhost specificity and allow them to readily infect humans. Therefore,there is a need to assess whether the HA protein in these strains can,in fact, convert to a form that can readily infect humans, and a furtherneed to identify HA variants with such ability. There is a further needto understand the characteristics of HA proteins generally that allow orprohibit infection of different subjects, particularly humans. There isalso a need for vaccines and therapeutic strategies for effectivetreatment or delay of onset of disease caused by influenza virus.

SUMMARY

The present invention binding agents with particular glycan bindingcharacteristics. In particular, the present invention provides bindingagents that bind to sialylated glycans having an umbrella-like topology.In some embodiments, binding agents in accordance with the inventionbind to umbrella-topology glycans with high affinity and/or specificity.In some embodiments, binding agents in accordance with the inventionshow a binding preference for umbrella-topology glycans as compared withcone-topology glycans. In some embodiments, binding agents in accordancewith the invention compete with hemagglutinin for binding to glycans onhemagglutinin receptors. In some embodiments, binding agents inaccordance with the invention compete with hemagglutinin for binding toumbrella-topology glycans.

The present invention also provides diagnostic and therapeutic reagentsand methods associated with provided binding agents, including vaccines.

The present invention particularly encompasses the recognition that HApolypeptide variants (e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10,H11, H12, H13, H14, H15, or H16 HA polypeptide variants) with alteredglycosylation can show increased (or decreased) binding to human HAreceptors as compared with a reference HA polypeptide. In someembodiments, the reference polypeptide is an HA polypeptide of any ofthe following:

A/South Carolina/1/18 (H1): (SEQ ID NO: 43)MEARLLVLLCAFAATNADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCKLKGIAPLQLGKCNIAGWLLGNPECDLLLTASSWSYIVETSNSENGTCYPGDFIDYEELREQLSSVSSFEKFEIFPKTSSWPNHETTKGVTAACSYAGASSFYRNLLWLTKKGSSYPKLSKSYVNNKGKEVLVLWGVHHPPTGTDQQSLYQNADAYVSVGSSKYNRRFTPEIAARPKVRDQAGRMNYYWTLLEPGDTITFEATGNLIAPWYAFALNRGSGSGIITSDAPVHDCNTKCQTPHGAINSSLPFQNIHPVTIGECPKYVRSTKLRMATGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAIDGITNKVNSVIEKMNTQFTAVGKEFNNLERRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVRNLYEKVKSQLKNNAKEIGNGCFEFYHKCDDACMESVRNGTYDYPKYSEESKLNREEIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAI SFWMCSNGSLQCRICIA/Brisbane/59/07 (H1): (SEQ ID NO: 44)MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENSHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGESSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQKALYHTENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWILLEPGDTIIFEANGNLIAPRYAFALSRGFGSGIINSNAPMDKCDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLERRMENLNKKVDDGFIDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAIS FWMCSNGSLQCRICIA/California/04/09 (H1): (SEQ ID NO: 45)MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADTYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAI SFWMCSNGSLQCRICIA/Albany/6/58 (H2): (SEQ ID NO: 46)MAIIYLILLFTAVRGDQICIGYHANNSTEKVDTILERNVTVTHAKDILEKTHNGKLCKLNGIPPLELGDCSIAGWLLGNPECDRLLSVPEWSYIMEKENPRDGLCYPGSFNDYEELKHLLSSVKHFEKVKILPKDRWTQHTTTGGSRACAVSGNPSFFRNMVWLTKKGSNYPVAKGSYNNTSGEQMLIIWGVHHPNDETEQRTLYQNVGTYVSVGTSTLNKRSTPDIATRPKVNGLGSRMEFSWTLLDMWDTINFESTGNLIAPEYGFKISKRGSSGIMKTEGTLGNCETKCQTPLGAINTTLPFHNVHPLTIGECPKYVKSEKLVLATGLRNVPQIESRGLFGAIAGFIEGGWQGMVDGWYGYHHSNDQGSGYAADKESTQKAFDGITNRVNSVIEKMNTQFEAVGKEFSNLERRLENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVKMQLRDNVKELGNGCFEFYPKCDDECMNSVKNGTYDYPKYEEESKLNRNEIKGVKLSSMGVYQILAIYATVAGSLSLAIMMAGISFWM CSNGSLQCRICIA/Aichi/1/68 (H3): (SEQ ID NO: 47)MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGIHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENAEEMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVVLLGF IMWACQRGNIRCNICIA/Moscow/10/99 (H3): (SEQ ID NO: 48)MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTLVKTITNDQIEVTNATELVQSSSTGRICDSPHQILDGENCTLIDALLGDPHCDGFQNKEWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVAQNGTSSSCKRRSIKSFFSRLNWLHQLKYRYPALNVTMPNNDKFDKLYIWGVHHPSTDSDQTSLYTQASGRVTVSTKRSQQTVIPNIGSRPWVRGISSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMMDGWYGFRHQNSEGTGQAADLKSTQAAINQINGKLNRLIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTRKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVVLLGF IMWACQKGNIRCNICIA/Perth/16/09 (H3): (SEQ ID NO: 49)MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGEICDSPHQILDGKNCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSKNSFFSRLNWLTHLNFKYPALNVTMPNNEQFDKLYIWGVHHPGTDKDQIFLYAQASGRITVSTKRSQQTVSPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGF IMWACQKGNIRCNICIA/Vietnam/1203/04 (H5): (SEQ ID NO: 50)MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVINKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVA GLSLWMCSNGSLQCRICIA/Egypt/2786-NAMRU3/06 (H5): (SEQ ID NO: 51)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGRSSFFRNVVWLIKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVINKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVA GLFLWMCSNGSLQCRICIA/New York/107/03 (H7): (SEQ ID NO: 52)MNTQILAFIACVLIGVKGDKICLGHHAVANGTKVNTLTERGIEVVNATETVETTNIKKICTQGKRPTDLGQCGLLGTLIGPPQCDQFLEFSSDLIIERREGTDICYPGRFTNEESLRQILRRSGGIGKESMGFTYSGIRTNGATSACTRSGSSFYAEMKWLLSNSDNAAFPQMTKAYRNPRNKPALIIWGVHHSESVSEQTKLYGSGNKLITVRSSKYQQSFTPNPGARRIDFHWLLLDPNDTVTFTFNGAFIAPDRTSFFRGESLGVQSDAPLDSSCRGDCFHSGGTIVSSLPFQNINSRTVGKCPRYVKQKSLLLATGMRNVPEKPKPRGLFGAIAGFIENGWEGLINGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIGKTNQQFELIDNEFNEIEQQIGNVINWTRDAMTEIWSYNAELLVAMENQHTIDLADSEMSKLYERVKKQLRENAEEDGTGCFEIFHKCDDQCMESIRNNTYDHTQYRTESLQNRIQIDPVKLSSGYKDIILWFSFGASCFLLLAIAMGLVFICIKNGNMQCTI CI

The present invention also particularly encompasses the recognition thatHA polypeptide variants (e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10,H11, H12, H13, H14, H15, or H16 HA polypeptide variants) withalterations in the HA loop region, can show increased (or decreased)binding to human HA receptors as compared with a reference HApolypeptide (including, for example, an HA polypeptide of any of SEQ IDNOs: 43-52).

In some embodiments, the present invention encompasses the recognitionthat H5 HA polypeptide variants with altered glycosylation can showincreased (or decreased) binding to human HA receptors as compared witha reference HA polypeptide. In some embodiments, the referencepolypeptide is an HA polypeptide of any of the following:

A/Hongkong/486/97 (SEQ ID NO: 53)MEKIVLLLATVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILERTHNGKLCDLNGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSNHDASSGVSSACPYLGRSSFFRNVVWLIKKNSAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPEIATRPKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNTPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIINKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVKNGTYDYPQYSEEARLNREEISGVKLESMGTYQILSIYSTVASSLALAIMVA GLSLWMCSNGSLQCRICIA/Hongkong/213/03 (SEQ ID NO: 54)MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKNSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVA GLSLWMCSNGSLQCRICIA/Vietnam/1203/04 (SEQ ID NO: 205)MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVA GLSLWMCSNGSLQCRICIA/Indonesia/5/05 (SEQ ID NO: 55)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTMLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRESRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESIRNGTYNYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMMA GLSLWMCSNGSLQCRICIA/Egypt/2786-NAMRU3/06 (SEQ ID NO: 206)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGRSSFFRNVVWLIKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVA GLFLWMCSNGSLQCRICI

The present invention also particularly encompasses the recognition thatH5 HA polypeptide variants with alterations in the HA loop region, canshow increased (or decreased) binding to human HA receptors as comparedwith a reference HA polypeptide (including, for example, an HApolypeptide of any of SEQ ID NO: 50, 51, and 53-55, 205 and 206).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Alignment of exemplary sequences of wild type HA. Sequenceswere obtained from the NCBI influenza virus sequence database (availablethrough the world wide web at ncbi.nlm.nih.gov/genomes/FLU/FLU). H1_Av(SEQ ID NO: 1). H1_Hu1 (SEQ ID NO: 2). H1_Hu2 (SEQ ID NO: 3). H2_Av (SEQID NO: 4). H2_Hu (SEQ ID NO: 5). H3_Av (SEQ ID NO: 6). H3_Hu1 (SEQ IDNO: 7). H3_Hu2 (SEQ ID NO: 8). H4_Av (SEQ ID NO: 9). H5_Av1 (SEQ ID NO:10). H5_Av2 (SEQ ID NO: 11). H6_Av (SEQ ID NO: 12). H7_Av (SEQ ID NO:13). H8_Av (SEQ ID NO: 14). H9_Av (SEQ ID NO: 15). H10_Av (SEQ ID NO:16). H11_Av (SEQ ID NO: 17). H12_Av (SEQ ID NO: 18). H13_Av (SEQ ID NO:19). H14_Av (SEQ ID NO: 20). H15_Av (SEQ ID NO: 21). H16_Av (SEQ ID NO:22).

FIGS. 2A-B. Sequence alignment of HA glycan binding domain. Gray:conserved amino acids involved in binding to sialic acid. Red:particular amino acids involved in binding to Neu5Acα2-3/6Gal motifs.Yellow: amino acids that influence positioning of Q226 (137, 138) andE190 (186, 228). Green: amino acids involved in binding to othermonosaccharides (or modifications) attached to Neu5Acα2-3/6Gal motif.The sequence for ASI30, APR34, ADU63, ADS97 and Viet04 were obtainedfrom their respective crystal structures. The other sequences wereobtained from SwissProt (us.expasy.org). Abbreviations: ADA76,A/duck/Alberta/35/76 (H1N1) (SEQ ID NO: 23); ASI30, A/Swine/Iowa/30(H1N1) (SEQ ID NO: 24); APR34, A/Puerto Rico/8/34 (H1N1) (SEQ ID NO:25); ASC18, A/South Carolina/1/18 (H1N1) (SEQ ID NO: 26); AT91,A/Texas/36/91 (H1N1) (SEQ ID NO: 27); ANY18, A/New York/1/18 (H1N1) (SEQID NO: 28); ADU63, A/Duck/Ukraine/1/63 (H3N8) (SEQ ID NO: 29); AAI68,A/Aichi/2/68 (H3N2) (SEQ ID NO: 30); AM99, A/Moscow/10/99 (H3N2) (SEQ IDNO: 31); ADS97, A/Duck/Singapore/3/97 (H5N3) (SEQ ID NO: 32); Viet04,A/Vietnam/1203/2004 (H5N1) (SEQ ID NO: 33).

FIGS. 3A-B. Sequence alignment illustrating conserved subsequencescharacteristic of H1 HA. FIG. 3A presents the same alignment that waspresented in FIG. 1A, except that FIG. 3A indicates the presence of anadditional conserved subsequence. FIG. 3B presents the same alignmentthat was presented in FIG. 1C, except that FIG. 3A indicates thepresence of an additional conserved subsequence. FIG. 3A discloses SEQID NOS 208-229, respectively, in order of appearance. FIG. 3B disclosesSEQ ID NOS 230-251, respectively, in order of appearance.

FIGS. 4A-B. Sequence alignment illustrating conserved subsequencescharacteristic of H3 HA. FIG. 4A presents the same alignment that waspresented in FIG. 1A, except that FIG. 4A indicates the presence of anadditional conserved subsequence. FIG. 4B presents the same alignmentthat was presented in FIG. 1C, except that FIG. 4A indicates thepresence of an additional conserved subsequence. FIG. 4A discloses SEQID NOS 252-271, respectively, in order of appearance. FIG. 4B disclosesSEQ ID NOS 271-295, respectively, in order of appearance.

FIG. 5A-1. Sequence alignment illustrating conserved subsequencescharacteristic of H5 HA. FIG. 5A-1 presents the same alignment that waspresented in FIG. 1A, except that FIG. 5A-1 indicates the presence of anadditional conserved subsequence. FIG. 5A-1 discloses SEQ ID NOS296-317, respectively, in order of appearance.

FIG. 5A-2 presents the same alignment that was presented in FIG. 1C,except that FIG. 5A-2 indicates the presence of an additional conservedsubsequence. FIG. 5A-2 discloses SEQ ID NOS 318-339, respectively, inorder of appearance.

FIGS. 5B1-B5 present additional H5 HA sequence alignments. Consensus(SEQ ID NO: 34); AAL59142 (SEQ ID NO: 35); AAZ29963 (SEQ ID NO: 36);ABA70758 (SEQ ID NO: 37); ABB87042 (SEQ ID NO: 38); ABD14810 (SEQ ID NO:39); ABD46740 (SEQ ID NO: 40); ABD85144 (SEQ ID NO: 41); and ABE97569(SEQ ID NO: 42).

FIG. 5B-6 shows additional avian 4 (HA) H5N1 viral strains.

FIGS. 6A to 6C-F. Framework for understanding glycan receptorspecificity. α2-3- and/or α2-6-linked glycans can adopt differenttopologies. According to the present invention, the ability of an HApolypeptide to bind to certain of these topologies confers upon it theability to mediate infection of different hosts, for example, humans. Asillustrated in FIG. 6A, the present invention defines two particularlyrelevant topologies, a “cone” topology and an “umbrella” topology. Thecone topology can be adopted by α2-3- and/or α2-6-linked glycans, and istypical of short oligosaccharides or branched oligosaccharides attachedto a core (although this topology can be adopted by certain longoligosaccharides). The umbrella topology can only be adopted byα2-6-linked glycans (presumably due to the increased conformationalplurality afforded by the extra C5-C6 bond that is present in the α2-6linkage), and is predominantly adopted by long oligosaccharides orbranched glycans with long oligosaccharide branches, particularlycontaining the motif Neu5Acα2-6Galβ1-3/4GlcNAc-. As described herein,ability of HA polypeptides to bind the umbrella glycan topology, confersbinding to human receptors and/or ability to mediate infection ofhumans. FIGS. 6B-1 to 6B-2 specifically show the topology of α2-3 andα2-6 as governed by the glycosidic torsion angles of the trisaccharidemotifs—Neu5Acα2-3Galβ1-3/4GlcNAc and Neu5Acα2-6Galβ1-4GlcNAcrespectively. A parameter (θ)—angle between C2 atom of Neu5Ac and C1atoms of the subsequent Gal and GlcNAc sugars in these trisaccharidemotifs was defined to characterize the topology. Superimposition of theθ contour and the conformational maps of the α2-3 and α2-6 motifs showsthat α2-3 motifs adopt 100% cone-like topology and α2-6 motifs sampledboth cone-like and umbrella-like topologies (FIGS. 6C-A to 6C-F). In thecone-like topology sampled by α2-3 and α2-6, GlcNAc and subsequentsugars are positioned along a region spanning a cone. Interactions of HAwith cone-like topology primarily involve contacts of amino acids at thenumbered positions (based on H3 HA numbering) with Neu5Ac and Galsugars. On the other hand, in umbrella-like topology, which is unique toα2-6, \ GlcNAc and subsequent sugars bend towards the HA binding site(as observed in HA-α2-6 co-crystal structures). Longer α2-6oligosaccharides (e.g. at least a tetrasaccharide) would favor thisconformation since it is stabilized by intra-sugar van der Waals contactbetween acetyl groups of GlcNAc and Neu5Ac. HA interactions withumbrella-like topology involve contacts of amino acids at the numberedpositions (based on H3 HA numbering) with GlcNAc and subsequent sugarsin addition to contacts with Neu5Ac and Gal sugars. FIGS. 6C-A through6C-F depict conformational sampling of cone- and umbrella-like topologyby α2-3 and α2-6. FIGS. 6C-A through 6C-D show the conformational (ϕ, ψ)maps of Neu5Acα2-3Gal, Neu5Acα2-6Gal, Galβ1-3GlcNAc, and Galβ1-4GlcNAclinkages, respectively. These maps obtained from GlycoMaps DB (availablethrough the world wide web at glycosciences.de/modeling/glycomapsdb/)were generated using ab initio MD simulations using MM3 force field.Energy distribution is color coded starting from dark (representinghighest energy) to light representing lowest energy. Encircled regions1-5 represent (ϕ,ψ) values observed for the α2-3 and α2-6oligosaccharides in the HA-glycan co-crystal structures. The transconformation (encircled region 1) of Neu5Acα2-3Gal predominates in HAbinding pocket with the exception of the co-crystal structure ofA/Aichi/2/68 H3N2 HA with α2-3 where this conformation is gauche(encircled region 2). On the other hand, the cis conformation ofNeu5Acα2-6Gal (encircled region 3) predominates in HA binding pocket.The cone-like topology is sampled by encircled regions 1 and 2 and theumbrella-like topology is sampled by encircled region 3. FIGS. 6C-Ethrough 6C-F show sampling of cone-like and umbrella-like topologies byα2-3 and α2-6 motifs, respectively. The darker regions in theconformational maps were used as the outer boundaries to calculate the θparameter (angle between C2 atom of Neu5Ac and C1 atoms of subsequentGal and GlcNAc sugars) for a given set of (ϕ,ψ) values. Based on theenergy cutoff, the value of θ>110° was used to characterize cone-liketopology and θ<100° was used to characterize umbrella-like topology.Superimposition of the θ contour with the conformational energy mapindicated that α2-3 motif adopts 100% cone-like topology since it wasenergetically unfavorable to adopt umbrella-like topology. On the otherhand, the α2-6 motif sampled both the cone-like and umbrella-liketopologies and this sampling was classified based on the ω angle(O—C6-C5-H5) of Neu5Acα2-6Gal linkage.

FIG. 7. Interactions of HA residues with cone vs. umbrella glycantopologies. Analysis of HA-glycan co-crystals reveals that the positionof Neu5Ac relative to the HA binding site is almost invariant. Contactswith Neu5Ac involve highly conserved residues such as F98, S/T136, W153,H183 and L/I194. Contacts with other sugars involve different residues,depending on whether the sugar linkage is α2-3 or α2-6 and whether theglycan topology is cone or umbrella. For example, in the cone topology,the primary contacts are with Neu5Ac and with Gal sugars. E190 and Q226play particularly important roles in this binding. This Figure alsoillustrates other positions (e.g., 137, 145, 186, 187, 193, 222) thatcan participate in binding to cone structures. In some cases, differentresidues can make different contacts with different glycan structures.The type of amino acid in these positions can influence ability of an HApolypeptide to bind to receptors with different modification and/orbranching patterns in the glycan structures. In the umbrella topology,contacts are made with sugars beyond Neu5Ac and Gal. This Figureillustrates residues (e.g., 137, 145, 156, 159, 186, 187, 189, 190, 192,193, 196, 222, 225, 226) that can participate in binding to umbrellastructures. In some cases, different residues can make differentcontacts with different glycan structures. The type of amino acid inthese positions can influence ability of an HA polypeptide to bind toreceptors with different modification and/or branching patterns in theglycan structures. In some embodiments, a D residue at position 190and/or a D residue at position 225 contribute(s) to binding to umbrellatopologies.

FIG. 8. Exemplary cone topologies. This Figure illustrates certainexemplary (but not exhaustive) glycan structures that adopt conetopologies.

FIGS. 9A-1 to 9B. Exemplary umbrella topologies. (A) Certain exemplary(but not exhaustive) N- and O-linked glycan structures that can adoptumbrella topologies. (B) Certain exemplary (but not exhaustive) O-linkedglycan structures that can adopt umbrella topologies.

FIGS. 10A-B. Glycan profile of human bronchial epithelial cells andhuman colonic epithelial cells. To further investigate the glycandiversity in the upper respiratory tissues, N-linked glycans wereisolated from HBEs (a representative upper respiratory cell line) andanalyzed using MALDI-MS. The predominant expression of α2-6 in HBEs wasconfirmed by pre-treating the sample with Sialidase S (α2-3 specific)and Sialidase A (cleaves and SA). The predominant expression of glycanswith long branch topology is supported by TOF-TOF fragmentation analysisof representative mass peaks. To provide a reference for glycandiversity in the upper respiratory tissues, the N-linked glycan profileof human colonic epithelial cells (HT29; a representative gut cell line)was obtained. This cell line was chosen because the current H5N1 viruseshave been shown to infect gut cells. Sialidase A and S pre-treatmentcontrols showed predominant expression of α2-3 glycans in the HT-29cells. Moreover, the long branch glycan topology is not as prevalent asobserved for HBEs. Therefore, human adaptation of the H5N1 HA wouldinvolve HA mutations that would enable high affinity binding to thediverse glycans expressed in the human upper respiratory tissues (e.g.,umbrella glycans).

FIGS. 11A-D. Conformational map and solvent accessibility ofNeu5Acα2-3Gal and Neu5Acα2-6Gal motifs. FIG. 11-A shows theconformational map of Neu5Acα2-3Gal linkage. The encircled region 2 isthe trans conformation observed in the APR34_H1_23, ADU63_H3_23 andADS97_H5_23 co-crystal structures. The encircled region 1 is theconformation observed in the AAI68_H3_23 co-crystal structure. FIG. 11-Bshows the conformational map of Neu5Acα2-6Gal where the cis-conformation(encircled region 3) is observed in all the HA-α2-6 sialylated glycanco-crystal structures. FIG. 11-C shows difference between solventaccessible surface area (SASA) of Neu5Ac α2-3 and α2-6 sialylatedoligosaccharides in the respective HA-glycan co-crystal structures. Thered and cyan bars respectively indicate that Neu5Ac in α2-6 (positivevalue) or α2-3 (negative value) sialylated glycans makes more contactwith glycan binding site. FIG. 11-D shows difference between SASA ofNeuAc in α2-3 sialylated glycans bound to swine and human H1(H1_(α2-3)), avian and human H3 (H3_(α2-3)), and of NeuAc in α2-6sialylated glycans bound to swine and human H1 (H1_(α2-6)). The negativebar for H3_(α2-3) indicates lesser contact of the human H3 HA withNeu5Acα2-3Gal compared to that of avian H3. Torsion angles—ϕ: C2-C1-O—C3(for Neu5Acα2-3/6 linkage); ψ: C1-O—C3-H3 (for Neu5Acα2-3Gal) orC1-O—C6-C5 (for Neu5Acα2-6Gal); ω: O—C6-C5-H5 (for Neu5Acα2-6Gal)linkages. The ϕ, ψ maps were obtained from GlycoMaps DB (availablethrough the world wide web at glycosciences.de/modeling/glycomapsdb/)which was developed by Dr. Martin Frank and Dr. Claus-Wilhelm von derLieth (German Cancer Research Institute, Heidelberg, Germany). Thecoloring scheme from high energy to low energy is from bright red tobright green, respectively.

FIGS. 12A-B. Lectin staining of upper respiratory tissue sections. Aco-stain of the tracheal tissue with Jacalin (lighter) and ConA (darker)reveals a preferential binding of Jacalin (binds specifically toO-linked glycans) to goblet cells on the apical surface of the tracheaand ConA (binds specifically to N-linked glycans) to the ciliatedtracheal epithelial cells. Without wishing to be bound by any particulartheory, we note that this finding suggests that goblet cellspredominantly express O-linked glycans while ciliated epithelial cellspredominantly express N-linked glycans. Co-staining of trachea withJacalin and SNA (dark; binds specifically to α2-6) shows binding of SNAto both goblet and ciliated cells. On the other hand, co-staining ofJacalin (lighter) and MAL (darker), which specifically binds to α2-3sialylated glycans, shows weak minimal to no binding of MAL to thepseudostratified tracheal epithelium but extensive binding to theunderlying regions of the tissue. Together, the lectin staining dataindicated predominant expression and extensive distribution of α2-6sialylated glycans as a part of both N-linked and O-linked glycansrespectively in ciliated and goblet cells on the apical side of thetracheal epithelium.

FIG. 13: Comparison of RBS of H5N1 HAs across different genetic clades.A/Hong Kong/486/97, Genetic Clade 0 (HK_486_97_c0) (SEQ ID NO: 76);A/Duck/Hunan/795/02, Genetic Clade 2.1.1 (DK_Hunan_795_02_c.2.1.1) (SEQID NO: 77); A/Hong Kong/213/03, Genetic Clade 1 (Hk_213_03_c1) (SEQ IDNO: 78); A/Vietnam/1194/04, Genetic Clade 1 (Viet_1194_04_c1) (SEQ IDNO: 79); A/Vietnam/1203/04, Genetic Clade 1 (Viet_1203_04_c1) (SEQ IDNO: 80); A/Indonesia/5/05, Genetic Clade 2.1.3 (Ind_5_05_c2.1.3) (SEQ IDNO: 81); A/Anhui/1/05, Genetic Clade 2.3.4 (Anhui_1_05_c2.3.4) (SEQ IDNO: 82); A/Egypt/2786-NAMRU3/06, Genetic Clade 2.2(Egypt_2876-N3_06_c2.2) (SEQ ID NO: 83); A/goose/Guiyang/337/06, GeneticClade 4 (Go_Guiy_337_06_c4) (SEQ ID NO: 84); A/Egypt/2321-NAMRU3/07,Genetic Clade 2.2.1 (Egypt_2321-N3_07_c2.2.1) (SEQ ID NO: 85);A/Egypt/3300-NAMRU3/08, Genetic Clade 2.2.1 (Egypt_3300-N3_08_c.2.2.1)(SEQ ID NO: 86); A/common magpie/Hong Kong/5052/07, Genetic Clade 2.3.2(Mag_HK_5052_07_c.2.3.2) (SEQ ID NO: 87);A/Chicken/Vietnam/NCVD-016/2008, Genetic Clade 7(Ck_Viet_NCVD-016_08_c7) (SEQ ID NO: 88).

FIG. 14: Adapted from Stevens et al., 2008, J. Mol. Biol., 381:1382-94.Viet0304: A/Vietnam/1203/04. Ind505: A/Indonesia/5/05. LS: Q226L andG228S mutations RBS of HA.

FIG. 15: Viet0304-LS and Viet0304-RLS (equivalent to Ind505-LS) HAs wereanalyzed in a dose-dependent fashion on our glycan array comprising ofrepresentative avian and human receptors. Both mutants showed minimalα2-6 binding (binding signals observed only at high HA concentration),which contrasts with the high affinity α2-6 binding shared by humanadapted HAs.

FIG. 16: Dose dependent analysis of H5N1 HAs that lack glycosylation atN158 in the context of LS mutation. Removal of glycosylation at N158 inthe context of LS mutation increased human receptor binding affinity tothe same range (Kd′˜20 pM) as observed for human adapted H1N1 and H2N2HAs. Removal of glycosylation at N158 on H5 HA template with Q226Lmutation alone (i.e., without G228S of the LS mutation) showed improvedpreference to human receptors (over avian receptors), but substantiallylowered human receptor-binding affinity in comparison withN158-deglycosylated LS mutant.

FIG. 17: Comparing salient features of RBS between human adapted H2N2 HA(Alb6_58) and H5N1 (Viet1203_04) HA. Four differences in H2N2 HA ascompared with H5N1 HA include: (1) Composition of 130 loop in H2N2 HAwhich includes a deletion, (2) lack of glycosylation at 158 position inH2N2 HA, (3) amino acid composition of the base of the RBS involvingpositions 137, 221, 226 and 228, and (4) amino acid composition at topof the RBS involving positions 188, 192 and 193.

FIG. 18: Comparison of the RBS of the H5 HA mutants with that of H2N2HA. A1b6_58_H2N2 (SEQ ID NO: 89): A/Albany/6/58 H2N2 HA; Viet1203_04_D(SEQ ID NO: 90): modified version of A/Vietnam/1203/04 HA;Viet1203_04_D_H2RBS (SEQ ID NO: 91): mutant of Viet1203_04 D withdeletion at 130 and 13 amino acid substitutions; Viet1203_04_D_H2RBSmin(SEQ ID NO: 92): mutant of Viet1203_04_D with deletion at 130 and 7substitutions; ckEgy_07 (SEQ ID NO: 93): A/chicken/Egypt/R2/2007 H5N1 HAthat already has deletion at 130; ckEgy_07_H2RBS (SEQ ID NO: 94): mutantof ckEgy_07 with 8 substitutions; ckViet_08 (SEQ ID NO: 95):A/chicken/Vietnam/NCVD-093/2008 H5N1 HA that already has switch incharge at 192 and 193 positions; ckViet_08 H2RBS (SEQ ID NO: 96): mutantof ckViet_08 with deletion at 130 and 6 substitutions; ckViet_08H2RBSmin(SEQ ID NO: 97): mutant of ckViet_08 with deletion at 130 and 4substitutions. The residue positions that are substituted are in boldand highlighted. The deletion in 130 loop is shown in bold highlighted.Glycosylation at 158 is highlighted.

FIG. 19: Dose dependent analysis of mutant of H5N1 HA(Viet1203_04_D_H2RBS) designed such that the molecular composition ofits RBS mimics that of human-adapted H2N2 HA. This mutant shows highlyspecific high affinity binding to human receptors that is characteristicof other human adapted HAs. The binding affinity of this mutant to humanreceptor (6′SLN-LN) is quantified by Kd′˜3 pM that is in the same rangeas that of human adapted H1N1 and H2N2 HAs.

FIG. 20: Characteristic glycan-receptor binding properties of pandemicHAs The HAs from prototypic human-adapted pandemic 1918 H1N1 (S1) and1958 H2N2 (S2) and 2009 H1N1 (S3) strains show specific high affinitybinding to human receptors (6′SLN-LN) with minimal to substantiallylower affinity binding (relative to human receptor affinity) to avianreceptors (3′SLN-LN). On the other hand introducing the hallmark LSmutation in Viet1203_04 H5N1 HA sequence does not switch its receptorpreference to the human receptor binding seen with the pandemic HAs.

FIG. 21: Phylogeny tree of representative sequences from HA subtypes

Branches leading to clade 1 & 2 HAs are labeled and colored in red andblue, respectively. Closely related subtypes are located on branchesclose to one another.

FIG. 22: Key structural features within the RBS of H5 HA

Shown in the figure is the cartoon rendering of RBS of Alb6_58 HA (gray)and Viet0304 (green) with side chains of amino acids that are differentbetween these HAs. The four features that distinguish the H2 and H5 RBSas described in the text are highlighted in dotted red circles.

FIG. 23: Sequence alignment of RBS of representative H2 and H5 HAs

HA sequences from the pandemic H2N2 strain (A/Albany/6/58 or Alb58) (SEQID NO: 98), representative human isolates from 1997-2006 (A/HongKong/486/97 or HK_486_97 (SEQ ID NO: 105), A/Hong Kong/213/03 orHK_213_03 (SEQ ID NO: 104), A/Vietnam/1203/04 or Viet1203_04 (SEQ ID NO:103), A/Indonesia/5/05 or Ind_5_05 (SEQ ID NO: 102), A/Egypt/2786-NAMRU3or Egy_2786-N3_06 (SEQ ID NO: 101)) along with the chosen H5 HA template(A/chicken/Egypt/R2/07 or ckEgy_07 (SEQ ID NO: 99)) for introducing LSmutation (ckEgy_07mutv5.3 (SEQ ID NO: 100)) are aligned.

FIGS. 24A-C: Glycan receptor-binding properties of ckEgy_07 and ckEgy_07harboring LS amino acid changes

Dose-dependent direct glycan-binding of HAs were performed on a glycanarray platform comprising of representative human and avian receptors.FIG. 24-A, the wild-type ckEgy_07 HA shows the typical specific and highaffinity avian-receptor binding characteristic of other wild-type H5N1HAs. FIG. 24-B, Introduction of the LS mutations on this HAquantitatively shifts its specificity to human receptor (6′SLN-LN) andsubstantially reducing its avian receptor binding to a minimal level.FIG. 24-C, binding of ckEgy_07 LS mutant to physiological humanreceptors expressed predominantly on apical surface of human trachealtissue section. The human receptor specificity and affinity from thedose-dependent binding profile together with the human tracheal tissuestaining of the mutant HA is such that it may be sufficient to conferaerosol transmission of H5N1 virus harboring this mutant HA in thecontext of other required changes (such as PB2).

FIGS. 25A-D: Analysis of effect of loss of glycosylation at the 158position in the context of LS mutations on glycan receptor binding of H5HA

FIG. 25-A, the ckEgy_07 LS mutant shows a quantitative human receptorswitch resembling other pandemic HAs. FIG. 25-B, the binding curve forA/California/04/2009 H1N1 HA adapted from previous study (S3) is shownfor comparison. FIG. 25-C, the T160A mutation in Viet03_04_ALS mutantremoves glycosylation sequon at N158 leading to loss of glycosylation atthis site. Although this mutant shows dramatic improvement in humanreceptor-binding, it retains most of its avian receptor-binding that isnot characteristic of pandemic HAs and the ckEgy_07_LS mutant (in thetop panel). FIG. 25-D, LS amino acid mutations introduced in Egy_06_HAsequence that naturally lacks glycosylation at 158 also shows the samebinding profile as Viet03_04_ALS mutant.

FIG. 26: Human-adaptive amino acid changes on H5N1 HA sequence thatnaturally acquired feature 2

A/chickenNietnam/NCVD-093/2008 avian H5N1 HA already acquired amino acidchanges in 190-helix where 192 position typically comprising of Thr hasmutated to Lys and 193 position typically comprising of Lys/Arg hasmutated to Met. Introducing 6 amino acid changes and a deletion to matchfeature 1 (deletion in 130-loop+A130T) and feature 3(S137R/S221P/Q226L/S227G/G228S) resulted in a mutant HA thatquantitatively switched its preference to human receptors even in thepresence of glycosylation at 158. However, introduction of the T160Aloss of glycosylation change along with LS, without the 130-loopdeletion results in dramatic reduction in binding to human and avianreceptors (data not shown). Therefore, for these H5N1 HAs, the deletionin 130-loop is a more critical change than the loss of glycosylation inthe context of LS mutation.

FIGS. 27A-B: Emergence of key features in recent avian H5 and human H5N1isolates

FIG. 27-A, Percentage of avian and human H5N1 isolates whose HA hasacquired amino acid changes to match features 1 and 4 of H2 HA RBS isplotted as function of year when the strain was isolated. There has beena dramatic increase in percentage of isolates having these key featuressince their initial emergence in 2007. Phylogenetic analysis of thesequences of these isolates showed that they belonged to clade 2.2.1.FIG. 27-B, Percentage of avian and human isolates whose HA has acquiredamino acid changes to match feature 2 of H2 HA RBS. Only a smallpercentage of H5N1 isolates have acquired this feature. Full-lengthnon-redundant HA sequences from NCBI Influenza Virus Resource werealigned, and number of occurrences of each of the features wascalculated in a given year and expressed as percentage. A total of 2277full-length non-redundant H5N1 sequences were employed for the analysis.

FIG. 28: Expanded nomenclature of glycans used in the glycan array

Neu5Ac: N-acetyl D-neuraminic acid; Gal: D-galactose; GlcNAc: N-acetylD-glucosamine. α/β: anomeric configuration of the pyranose sugars. Allthe sugars are linked via a spacer to biotin (-Sp-LC-LC-Biotin asdescribed in, and available through the world wide web at,functionalglycomics.org/static/consortium/resources/resourcecored5).

DESCRIPTION OF HA SEQUENCE ELEMENTS HA Sequence Element 1

HA Sequence Element 1 is a sequence element corresponding approximatelyto residues 97-185 (where residue positions are assigned using H3 HA asreference) of many HA proteins found in natural influenza isolates. Thissequence element has the basic structure:

(SEQ ID NO. 106) C (Y/F) P X₁ C X₂ W X₃ W X₄ H H P,wherein:

-   -   X₁ is approximately 30-45 amino acids long;    -   X₂ is approximately 5-20 amino acids long;    -   X₃ is approximately 25-30 amino acids long; and    -   X₄ is approximately 2 amino acids long.

In some embodiments, X₁ is about 35-45, or about 35-43, or about 35, 36,37, 38, 38, 40, 41, 42, or 43 amino acids long. In some embodiments, X₂is about 9-15, or about 9-14, or about 9, 10, 11, 12, 13, or 14 aminoacids long. In some embodiments, X₃ is about 26-28, or about 26, 27, or28 amino acids long. In some embodiments, X₄ has the sequence (G/A)(I/V). In some embodiments, X₄ has the sequence GI; in some embodiments,X₄ has the sequence GV; in some embodiments, X₄ has the sequence AI; insome embodiments, X₄ has the sequence AV. In some embodiments, HASequence Element 1 comprises a disulfide bond. In some embodiments, thisdisulfide bond bridges residues corresponding to positions 97 and 139(based on the canonical H3 numbering system utilized herein).

In some embodiments, and particularly in H1 polypeptides, X₁ is about 43amino acids long, and/or X₂ is about 13 amino acids long, and/or X₃ isabout 26 amino acids long. In some embodiments, and particularly in H1polypeptides, HA Sequence Element 1 has the structure:

(SEQ ID NO. 107) C Y P X_(1A) Y (A/T) (A/S) C X₂ W X₃ W X₄ H H P,wherein:

-   -   X_(1A) is approximately 27-42, or approximately 32-42, or        approximately 32-40, or approximately 26-41, or approximately        31-41, or approximately 31-39, or approximately 31, 32, 33, 34,        35, 36, 37, 38, 39, or 40 amino acids long, and X₂-X₄ are as        above.

In some embodiments, and particularly in H1 polypeptides, HA SequenceElement 1 has the structure:

(SEQ ID NO. 108)C Y P X_(1A) T (A/T) (A/S) C X₂ W (I/L) (T/V) X_(3A) W X₄ H H P,wherein:

-   -   X_(1A) is approximately 27-42, or approximately 32-42, or        approximately 32-40, or approximately 32, 33, 34, 35, 36, 37,        38, 39, or 40 amino acids long,    -   X_(3A) is approximately 23-28, or approximately 24-26, or        approximately 24, 25, or 26 amino acids long, and X₂ and X₄ are        as above.

In some embodiments, and particularly in H1 polypeptides, HA SequenceElement 1 includes the sequence:

(SEQ ID NO. 109) Q L S S I S S F E K,typically within X₁, (including within X_(1A)) and especially beginningabout residue 12 of X₁ (as illustrated, for example, in FIGS. 1-3).

In some embodiments, and particularly in H3 polypeptides, X₁ is about 39amino acids long, and/or X₂ is about 13 amino acids long, and/or X₃ isabout 26 amino acids long.

In some embodiments, and particularly in H3 polypeptides, HA SequenceElement 1 has the structure:

(SEQ ID NO. 110) C Y P X_(1A) S (S/N)(A/S) C X₂ W X₃ W X₄ H H P,wherein:

-   -   X_(1A) is approximately 27-42, or approximately 32-42, or        approximately 32-40, or approximately 23-38, or approximately        28-38, or approximately 28-36, or approximately 28, 29, 30, 31,        32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids long, and        X₂-X₄ are as above.

In some embodiments, and particularly in H3 polypeptides, HA SequenceElement 1 has the structure:

(SEQ ID NO. 111)C Y P X_(1A) S (S/N)(A/S) C X₂ W L (T/H) X_(3A) W X₄ H H P,wherein:

-   -   X_(1A) is approximately 27-42, or approximately 32-42, or        approximately 32-40, or approximately 32, 33, 34, 35, 36, 37,        38, 39, or 40 amino acids long,    -   X_(3A) is approximately 23-28, or approximately 24-26, or        approximately 24, 25, or 26 amino acids long, and X₂ and X₄ are        as above.

In some embodiments, and particularly in H3 polypeptides, HA SequenceElement 1 includes the sequence:

(SEQ ID NO. 112) (L/I)(V/I) A S S G T L E F,typically within X₁ (including within X_(1A)), and especially beginningabout residue 12 of X₁ (as illustrated, for example, in FIGS. 1, 2 and4).

In some embodiments, and particularly in H5 polypeptides, X₁ is about 42amino acids long, and/or X₂ is about 13 amino acids long, and/or X₃ isabout 26 amino acids long.

In some embodiments, and particularly in H5 polypeptides, HA SequenceElement 1 has the structure:

(SEQ ID NO. 113) C Y P X_(1A) S S A C X₂ W X₃ W X₄ H H P,wherein:

-   -   X_(1A) is approximately 27-42, or approximately 32-42, or        approximately 32-40, or approximately 23-38, or approximately        28-38, or approximately 28-36, or approximately 28, 29, 30, 31,        32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids long, and        X₂-X₄ are as.

In some embodiments, and particularly in H5 polypeptides, HA SequenceElement 1 has the structure:

(SEQ ID NO. 114) C Y P X_(1A) S S A C X₂ W L I X₃ A W X₄ H H P,wherein:

-   -   X_(1A) is approximately 27-42, or approximately 32-42, or        approximately 32-40, or approximately 32, 33, 34, 35, 36, 37,        38, 39, or 40 amino acids long, and    -   X_(3A) is approximately 23-28, or approximately 24-26, or        approximately 24, 25, or 26 amino acids long, and X₂ and X₄ are        as above.

In some embodiments, and particularly in H5 polypeptides, HA SequenceElement 1 is extended (i.e., at a position corresponding to residues186-193) by the sequence:

(SEQ ID NO. 115) N D A A E X X (K/R)

In some embodiments, and particularly in H5 polypeptides, HA SequenceElement 1 includes the sequence:

(SEQ ID NO. 116) Y E E L K H L X S X X N H F E K,typically within X₁, and especially beginning about residue 6 of X₁ (asillustrated, for example, in FIGS. 1, 2, and 5).

HA Sequence Element 2

HA Sequence Element 2 is a sequence element corresponding approximatelyto residues 324-340 (again using a numbering system based on H3 HA) ofmany HA proteins found in natural influenza isolates. This sequenceelement has the basic structure:

(SEQ ID NO. 117) G A I A G F I EIn some embodiments, HA Sequence Element 2 has the sequence:

(SEQ ID NO. 118) P X₁G A I A G F I E,wherein:

-   -   X₁ is approximately 4-14 amino acids long, or about 8-12 amino        acids long, or about 12, 11, 10, 9 or 8 amino acids long. In        some embodiments, this sequence element provides the HA0        cleavage site, allowing production of HA1 and HA2.

In some embodiments, and particularly in H1 polypeptides, HA SequenceElement 2 has the structure:

(SEQ ID NO. 119) P S (I/V) Q S R X_(1A) G A I A G F I E,wherein:

-   -   X_(1A) is approximately 3 amino acids long; in some embodiments,        X_(1A) is G (L/I) F.

In some embodiments, and particularly in H3 polypeptides, HA SequenceElement 2 has the structure:

(SEQ ID NO. 120) P X K X T R X_(1A) G A I A G F I E,wherein:

-   -   X_(1A) is approximately 3 amino acids long; in some embodiments,        X_(1A) is G (L/I) F.

In some embodiments, and particularly in H5 polypeptides, HA SequenceElement 2 has the structure:

(SEQ ID NO. 121) P Q R X X X R X X R X_(1A) G A I A G F I E,wherein:

-   -   X_(1A) is approximately 3 amino acids long; in some embodiments,        X_(1A) is G (L/I) F.

Definitions

Affinity: As is known in the art, “affinity” is a measure of thetightness with a particular ligand (e.g., an HA polypeptide) binds toits partner (e.g., and HA receptor). Affinities can be measured indifferent ways.

Binding: It will be understood that the term “binding”, as used herein,typically refers to a non-covalent association between or among agents.In many embodiments herein, binding is addressed with respect toparticular glycans (e.g., umbrella topology glycans or cone topologyglycans). It will be appreciated by those of ordinary skill in the artthat such binding may be assessed in any of a variety of contexts. Insome embodiments, binding is assessed with respect to free glycans. Insome embodiments, binding is assessed with respect to glycans attached(e.g., covalently linked to) a carrier. In some such embodiments, thecarrier is a polypeptide. In some embodiments, binding is assessed withrespect to glycans attached to an HA receptor. In such embodiments,reference may be made to receptor binding or to glycan binding.

Binding agent: In general, the term “binding agent” is used herein torefer to any entity that binds to glycans (e.g., to umbrella-topologyglycans) as described herein. Binding agents may be of any chemicaltype. In some embodiments, binding agents are polypeptides (including,e.g., antibodies or antibody fragments); in some such embodiments,binding agents are HA polypeptides and/or variants thereof and/orcharacteristic portions thereof; in some embodiments, binding agents arepolypeptides whose amino acid sequence does not include an HAcharacteristic sequence (i.e., “Non-HA polypeptides”). In someembodiments, binding agents are small molecules. In some embodiments,binding agents are nucleic acids. In some embodiments, binding agentsare aptamers. In some embodiments, binding agents are polymers; in someembodiments, binding agents are non-polymeric. In some embodiments,binding agents are carbohydrates. In some embodiments, binding agentsare lectins. In some embodiments, binding agents as described hereinbind to sialylated glycans having an umbrella-like topology. In someembodiments, binding agents bind to umbrella-topology glycans with highaffinity and/or specificity. In some embodiments, binding agents show abinding preference for umbrella-topology glycans as compared withcone-topology glycans. In some embodiments, binding agents compete withhemagglutinin for binding to glycans on hemagglutinin receptors. In someembodiments, binding agents compete with hemagglutinin for binding toumbrella-topology glycans. In some embodiments, a binding agent providedherein is an umbrella topology blocking agent. In some embodiments, abinding agent provided herein is an umbrella topology specific blockingagent. In some embodiments, binding agents bind to umbrella topologyglycan mimics.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any agent that has activity in abiological system, and particularly in an organism. For instance, anagent that, when administered to an organism, has a biological effect onthat organism, is considered to be biologically active. In someembodiments, where a protein or polypeptide is biologically active, aportion of that protein or polypeptide that shares at least onebiological activity of the protein or polypeptide is typically referredto as a “biologically active” portion.

Characteristic portion: As used herein, the phrase a “characteristicportion” of a protein or polypeptide is one that contains a continuousstretch of amino acids, or a collection of continuous stretches of aminoacids, that together are characteristic of a protein or polypeptide.Each such continuous stretch generally will contain at least two aminoacids. Furthermore, those of ordinary skill in the art will appreciatethat typically at least 5, at least 10, at least 15, at least 20 or moreamino acids are required to be characteristic of a protein. In general,a characteristic portion is one that, in addition to the sequenceidentity specified above, shares at least one functional characteristicwith the relevant intact protein.

Characteristic sequence: A “characteristic sequence” is a sequence thatis found in all members of a family of polypeptides or nucleic acids,and therefore can be used by those of ordinary skill in the art todefine members of the family.

Cone topology: The phrase “cone topology” is used herein to refer to a3-dimensional arrangement adopted by certain glycans and in particularby glycans on HA receptors. As illustrated in FIG. 6, the cone topologycan be adopted by α2-3 sialylated glycans or by α2-6 sialylated glycans,and is typical of short oligonucleotide chains, though some longoligonucleotides can also adopt this conformation. The cone topology ischaracterized by the glycosidic torsion angles of Neu5Acα2-3Gal linkagewhich samples three regions of minimum energy conformations given by ϕ(C1-C2-O—C3/C6) value of about −60, about 60, or about 180 and(C2-O—C3/C6-H3/C5) samples −60 to 60 (FIGS. 11A-B). FIG. 8 presentscertain representative (though not exhaustive) examples of glycans thatadopt a cone topology.

Corresponding to: As used herein, the term “corresponding to” is oftenused to designate the position/identity of an amino acid residue in anHA polypeptide. Those of ordinary skill will appreciate that, forpurposes of simplicity, a canonical numbering system (based on wild typeH3 HA) is utilized herein (as illustrated, for example, in FIGS. 1-5),so that an amino acid “corresponding to” a residue at position 190, forexample, need not actually be the 190^(th) amino acid in a particularamino acid chain but rather corresponds to the residue found at 190 inwild type H3 HA; those of ordinary skill in the art readily appreciatehow to identify corresponding amino acids.

Degree of separation removed: As used herein, amino acids that are a“degree of separation removed” are HA amino acids that have indirecteffects on glycan binding. For example, one-degree-of-separation-removedamino acids may either: (1) interact with the direct-binding aminoacids; and/or (2) otherwise affect the ability of direct-binding aminoacids to interact with glycan that is associated with host cell HAreceptors; such one-degree-of-separation-removed amino acids may or maynot directly bind to glycan themselves. Two-degree-of-separation-removedamino acids either (1) interact with one-degree-of-separation-removedamino acids; and/or (2) otherwise affect the ability of theone-degree-of-separation-removed amino acids to interact withdirect-binding amino acids, etc.

Direct-binding amino acids: As used herein, the phrase “direct-bindingamino acids” refers to HA polypeptide amino acids which interactdirectly with one or more glycans that is associated with host cell HAreceptors.

Engineered: The term “engineered”, as used herein, describes apolypeptide whose amino acid sequence has been selected by man. Forexample, an engineered HA polypeptide has an amino acid sequence thatdiffers from the amino acid sequences of HA polypeptides found innatural influenza isolates. In some embodiments, an engineered HApolypeptide has an amino acid sequence that differs from the amino acidsequence of HA polypeptides included in the NCBI database.

HI polypeptide: An “H1 polypeptide”, as that term is used herein, is anHA polypeptide whose amino acid sequence includes at least one sequenceelement that is characteristic of H1 and distinguishes H1 from other HAsubtypes. Representative such sequence elements can be determined byalignments such as, for example, those illustrated in FIGS. 1-3 andinclude, for example, those described herein with regard to H1-specificembodiments of HA Sequence Elements.

H3 polypeptide: An “H3 polypeptide”, as that term is used herein, is anHA polypeptide whose amino acid sequence includes at least one sequenceelement that is characteristic of H3 and distinguishes H3 from other HAsubtypes. Representative such sequence elements can be determined byalignments such as, for example, those illustrated in FIGS. 1, 2, and 4and include, for example, those described herein with regard toH3-specific embodiments of HA Sequence Elements.

H5 polypeptide: An “H5 polypeptide”, as that term is used herein, is anHA polypeptide whose amino acid sequence includes at least one sequenceelement that is characteristic of H5 and distinguishes H5 from other HAsubtypes. Representative such sequence elements can be determined byalignments such as, for example, those illustrated in FIGS. 1, 2, and 5and include, for example, those described herein with regard toH5-specific embodiments of HA Sequence Elements.

HX polypeptide: An “HX polypeptide”, as that term is used herein, is anHA polypeptide whose amino acid sequence includes at least one sequenceelement that is characteristic of HX and distinguishes HX from other HAsubtypes, wherein “X” refers to the numbering of the HA subtype (e.g.,wherein when “X”=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or16, “HX”=H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,H15, or H16, respectively).

Hemagglutinin (HA) polypeptide: As used herein, the term “hemagglutininpolypeptide” (or “HA polypeptide’) refers to a polypeptide whose aminoacid sequence includes at least one characteristic sequence of HA. Awide variety of HA sequences from influenza isolates are known in theart; indeed, the National Center for Biotechnology Information (NCBI)maintains a database (available through the world wide web atncbi.nlm.nih.gov/genomes/FLU/flu) that, as of the filing of the presentapplication included 9796 HA sequences. Those of ordinary skill in theart, referring to this database, can readily identify sequences that arecharacteristic of HA polypeptides generally, and/or of particular HApolypeptides (e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12,H13, H14, H15, or H16 polypeptides); or of HAs that mediate infection ofparticular hosts, e.g., avian, camel, canine, cat, civet, environment,equine, human, leopard, mink, mouse, seal, stone martin, swine, tiger,whale, etc. For example, in some embodiments, an HA polypeptide includesone or more characteristic sequence elements found between aboutresidues 97 and about 185, about 324 and about 340, about 96 and about100, and/or about 130 and about 230 of an HA protein found in a naturalisolate of an influenza virus. In some embodiments, an HA polypeptidehas an amino acid sequence comprising at least one of HA SequenceElements 1 and 2, as defined herein. In some embodiments, an HApolypeptide has an amino acid sequence comprising HA Sequence Elements 1and 2, in some embodiments separated from one another by about 100 toabout 200, or by about 125 to about 175, or about 125 to about 160, orabout 125 to about 150, or about 129 to about 139, or about 129, about130, about 131, about 132, about 133, about 134, about 135, about 136,about 137, about 138, or about 139 amino acids. In some embodiments, anHA polypeptide has an amino acid sequence that includes residues atpositions within the regions 96-100 and/or 130-230 that participate inglycan binding. For example, many HA polypeptides include one or more ofthe following residues: Tyr98, Ser/Thr136, Trp153, His183, andLeu/Ile194. In some embodiments, an HA polypeptide includes at least 2,3, 4, or all 5 of these residues.

Isolated: The term “isolated”, as used herein, refers to an agent orentity that has either (i) been separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature or in an experimental setting); or (ii) produced by the handof man. Isolated agents or entities may be separated from at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, or more of the other components with which theywere initially associated. In some embodiments, isolated agents are morethan 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% pure.

Linkage Specific Blocking Agent (LSBA): As used herein, the term“linkage specific blocking agent” refers to an agent which binds to anHA receptor having an α2-6 sialylated glycan. In some embodiments, anLSBA selectively binds to an HA receptor having an α2-6 sialylatedglycan with at least about 40, 50, or 75% of the affinity of that for anHA receptor having an α2-3 sialylated glycan. In some embodiments, anLSBA selectively binds to an HA receptor having an α2-6 sialylatedglycan with at least about 2, 4, 5, or 10 times greater affinity thanthat for an HA receptor having an α2-3 sialylated glycan. In someembodiments, an LSBA has an affinity for an α2-6 sialylated glycan thatis at least 50, 100, 150, or 200% of its affinity for an α2-3 sialylatedglycan. In some embodiments, an LSBA may compete with hemagglutinin forbinding to an HA receptor. For example, an LSBA may selectively inhibitthe binding of an influenza virus particle (e.g., human or avianinfluenza virus) to an HA receptor based on the linkage characteristics(e.g., α2-6 sialylated glycan or α2-3 sialylated glycan). In someembodiments, an LSBA is a polypeptide. In some such embodiments, an LSBApolypeptide has an amino acid sequence that is substantially identicalor substantially homologous to that of a naturally-occurringpolypeptide. In some embodiments, an LSBA polypeptide is an HApolypeptide. In some embodiments, an LSBA polypeptide is anaturally-occurring HA polypeptide, or a fragment thereof. In someembodiments, an LSBA polypeptide has an amino acid sequence that is notrelated to that of an HA polypeptide. In some embodiments, an LSBApolypeptide is an antibody or fragment thereof. In some embodiments, anLSBA polypeptide is a lectin (e.g., SNA-1). In some embodiments, an LSBAis not a polypeptide. In some embodiments, an LSBA is a small molecule.In some embodiments, an LSBA is a nucleic acid.

Long oligosaccharide: For purposes of the present disclosure, anoligosaccharide is typically considered to be “long” if it includes atleast one linear chain that has at least four saccharide residues.

Non-natural amino acid: The phrase “non-natural amino acid” refers to anentity having the chemical structure of an amino acid (i.e.,:

and therefore being capable of participating in at least two peptidebonds, but having an R group that differs from those found in nature. Insome embodiments, non-natural amino acids may also have a second R grouprather than a hydrogen, and/or may have one or more other substitutionson the amino or carboxylic acid moieties.

Polypeptide: A “polypeptide”, generally speaking, is a string of atleast two amino acids attached to one another by a peptide bond. In someembodiments, a polypeptide may include at least 3-5 amino acids, each ofwhich is attached to others by way of at least one peptide bond. Thoseof ordinary skill in the art will appreciate that polypeptides sometimesinclude “non-natural” amino acids or other entities that nonetheless arecapable of integrating into a polypeptide chain, optionally.

Pure: As used herein, an agent or entity is “pure” if it issubstantially free of other components. For example, a preparation thatcontains more than about 90% of a particular agent or entity istypically considered to be a pure preparation. In some embodiments, anagent or entity is at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% pure.

Short oligosaccharide: For purposes of the present disclosure, anoligosaccharide is typically considered to be “short” if it has fewerthan 4, or certainly fewer than 3, residues in any linear chain.

Specificity: As is known in the art, “specificity” is a measure of theability of a particular ligand (e.g., an HA polypeptide) to distinguishits binding partner (e.g., a human HA receptor, and particularly a humanupper respiratory tract HA receptor) from other potential bindingpartners (e.g., an avian HA receptor).

Substantial homology: The phrase “substantial homology” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially homologous” ifthey contain homologous residues in corresponding positions. Homologousresidues may be identical residues. Alternatively, homologous residuesmay be non-identical residues will appropriately similar structuraland/or functional characteristics. For example, as is well known bythose of ordinary skill in the art, certain amino acids are typicallyclassified as “hydrophobic” or “hydrophilic” amino acids, and/or ashaving “polar” or “non-polar” side chains. Substitution of one aminoacid for another of the same type may often be considered a “homologous”substitution. Typical amino acid categorizations are summarized below:

Alanine Ala A nonpolar neutral 1.8 Arginine Arg R polar positive −4.5Asparagine Asn N polar neutral −3.5 Aspartic acid Asp D polar negative−3.5 Cysteine Cys C nonpolar neutral 2.5 Glutamic acid Glu E polarnegative −3.5 Glutamine Gln Q polar neutral −3.5 Glycine Gly G nonpolarneutral −0.4 Histidine His H polar positive −3.2 Isoleucine Ile Inonpolar neutral 4.5 Leucine Leu L nonpolar neutral 3.8 Lysine Lys Kpolar positive −3.9 Methionine Met M nonpolar neutral 1.9 PhenylalaninePhe F nonpolar neutral 2.8 Proline Pro P nonpolar neutral −1.6 SerineSer S polar neutral −0.8 Threonine Thr T polar neutral −0.7 TryptophanTrp W nonpolar neutral −0.9 Tyrosine Tyr Y polar neutral −1.3 Valine ValV nonpolar neutral 4.2

Ambiguous Amino Acids 3-Letter 1-Letter Asparagine or aspartic acid AsxB Glutamine or glutamic acid Glx Z Leucine or Isoleucine Xle JUnspecified or unknown amino acid Xaa XAs is well known in this art, amino acid or nucleic acid sequences maybe compared using any of a variety of algorithms, including thoseavailable in commercial computer programs such as BLASTN for nucleotidesequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acidsequences. Exemplary such programs are described in Altschul, et al.,Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990;Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLASTand PSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al.,Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins,Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods andProtocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999;all of the foregoing of which are incorporated herein by reference. Inaddition to identifying homologous sequences, the programs mentionedabove typically provide an indication of the degree of homology. In someembodiments, two sequences are considered to be substantially homologousif at least 50%, at least 55%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or more of their correspondingresidues are homologous over a relevant stretch of residues. In someembodiments, the relevant stretch is a complete sequence. In someembodiments, the relevant stretch is at least 10, at least 15, at least20, at least 25, at least 30, at least 35, at least 40, at least 45, atleast 50, at least 55, at least 60, at least 65, at least 70, at least75, at least 80, at least 85, at least 90, at least 95, at least 100, atleast 125, at least 150, at least 175, at least 200, at least 225, atleast 250, at least 275, at least 300, at least 325, at least 350, atleast 375, at least 400, at least 425, at least 450, at least 475, atleast 500 or more residues.

Substantial identity: The phrase “substantial identity” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially identical” ifthey contain identical residues in corresponding positions. As is wellknown in this art, amino acid or nucleic acid sequences may be comparedusing any of a variety of algorithms, including those available incommercial computer programs such as BLASTN for nucleotide sequences andBLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplarysuch programs are described in Altschul, et al., Basic local alignmentsearch tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al.,Methods in Enzymology; Altschul, et al., “Gapped BLAST and PSI-BLAST: anew generation of protein database search programs”, Nucleic Acids Res.25:3389-3402, 1997; Baxevanis, et al., Bioinformatics: A Practical Guideto the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al.,(eds.), Bioinformatics Methods and Protocols (Methods in MolecularBiology, Vol. 132), Humana Press, 1999; all of the foregoing of whichare incorporated herein by reference. In addition to identifyingidentical sequences, the programs mentioned above typically provide anindication of the degree of identity. In some embodiments, two sequencesare considered to be substantially identical if at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or more of their corresponding residues are identicalover a relevant stretch of residues. In some embodiments, the relevantstretch is a complete sequence. In some embodiments, the relevantstretch is at least 10, at least 15, at least 20, at least 25, at least30, at least 35, at least 40, at least 45, at least 50, at least 55, atleast 60, at least 65, at least 70, at least 75, at least 80, at least85, at least 90, at least 95, at least 100, at least 125, at least 150,at least 175, at least 200, at least 225, at least 250, at least 275, atleast 300, at least 325, at least 350, at least 375, at least 400, atleast 425, at least 450, at least 475, at least 500 or more residues.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refersto any agent that elicits a desired biological or pharmacologicaleffect.

Treatment: As used herein, the term “treatment” refers to any methodused to alleviate, delay onset, reduce severity or incidence, or yieldprophylaxis of one or more symptoms or aspects of a disease, disorder,or condition. For the purposes of the present invention, treatment canbe administered before, during, and/or after the onset of symptoms.

Umbrella topology: The phrase “umbrella topology” is used herein torefer to a 3-dimensional arrangement adopted by certain glycans and inparticular by glycans on HA receptors. The present invention encompassesthe recognition that binding to umbrella topology glycans ischaracteristic of HA proteins that mediate infection of human hosts. Asillustrated in FIG. 6A, the umbrella topology is typically adopted onlyby α2-6 sialylated glycans, and is typical of long (e.g., greater thantetrasaccharide) oligosaccharides. In some embodiments,umbrella-topology glycans are glycans exhibiting a three-dimensionalstructure substantially similar to the structure presented in FIG. 6A(right panel). In some embodiments, umbrella-topology glycans areglycans which contact HA polypeptides via the amino acid residues shownin FIG. 6A (right panel). In some embodiments, umbrella-topology glycansare glycans which are able to contact and/or specifically bind to theamino acid binding pocket shown in FIG. 6A (right panel). In someembodiments, glycan structural topology is classified based on parameterθ defined as angle between C₂ of Sia, C₁ of Gal, and C₁ of GlcNAc.Values of θ<100° represent cone-like topology adopted by α2-3 and shortα2-6 glycans. Values of θ>110° represent umbrella-like topology, such astopology adopted by long α2-6 glycans (FIGS. 6B-1 and 6B-2). An exampleof umbrella topology is given by ϕ angle of Neu5Acα2-6Gal linkage ofaround −60 (see, for example, FIGS. 11A-B). FIGS. 9A-9B present certainrepresentative (though not exhaustive) examples of glycans that canadopt an umbrella topology. The long α2-6 motifs presented in FIGS. 9A-1through 9A-7 includes Neu5Acα2-6 linked at the non-reducing end to along chain (e.g., at least a trisaccharide) found as a part ofbiological N-linked glycans, O-linked glycans, and glycolipids. Theboxed inset shows examples of the umbrella-topology long α2-6 glycanmoieties that are found as a part of biological glycans that bind tohigh affinity with HA. In some embodiments, umbrella-topology glycans(e.g., at a site) comprise a greater proportion of long (e.g. multiplelactosamine units) α2-6 oligosaccharide branches than short α2-6 (e.g.single lactosamine) branches. In some embodiments, umbrella-topologyglycans (e.g., at a site) comprise about 2-fold, about 3-fold, about4-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, orgreater than about 50-fold more long α2-6 oligosaccharide branches thanshort α2-6 (e.g. single lactosamine) branches. In some embodiments, theunique characteristic of HA interactions with umbrella-topology glycansand/or glycan decoys is the HA contact with a glycan comprising sialicacid (SA) and/or SA analogs at the non-reducing end. In someembodiments, chain length of the oligosaccharide is at least atrisaccharide (excluding the SA or SA analog). In some embodiments, acombination of the numbered residues shown in the right-hand panel ofFIG. 6A is involved in contacts with umbrella-like topology. In someembodiments, umbrella topology glycans are oligosaccharides of thefollowing form:

Neu5Acα2-6Sug1-Sug2-Sug3

where:

(a) Neu5Ac α2-6 is typically (but not essentially) at the non-reducingend;

(b) Sug1:

-   -   (i) is a hexose (frequently Gal or Glc) or hexosamine (GlcNAc or        GalNAc) in α or β configuration (frequently β- for N- and        O-linked extension and α- in the case of GalNAcα- that is        O-linked to glycoprotein);    -   (ii) no sugars other than Neu5Acα2-6 are attached to any of the        non-reducing positions of Sug1 (except when Sug1 is GalNAcα-        that is O-linked to the glycoprotein); and/or    -   (iii) non-sugar moieties such as sulfate, phosphate, guanidium,        amine, N-acetyl, etc. can be attached to non-reducing positions        (typically 6 position) of Sug1 (e.g., to improve contacts with        HA);

(c) Sug2 and/or Sug3 is/are:

-   -   (i) hexose (frequently Gal or Glc) or hexosamine (GlcNAc or        GalNAc) in α or β configuration (frequently β); and/or    -   (ii) sugars (such as Fuc) or non-sugar moieties such as sulfate,        phosphate, guanidium, amine, N-acetyl, etc. can be attached to        non-reducing positions of Sug2, Sug3, and/or Sug4;

(d) Linkage between any two sugars in the oligosaccharide apart fromNeu5Acα2-6 linkage can be 1-2, 1-3, 1-4, and/or 1-6 (typically 1-3 or1-4); and/or

(e) Structure where Neu5Acα2-6 is linked GalNAcα that is O-linked to theglycoprotein and additional sugars are linked to the non-reducing end ofGalNAcα for example

-   -   (i) Neu5Acα2-6(Neu5Acα2-3Galβ1-3)GalNAcα-    -   (ii) Neu5Acα2-6(Galβ1-3)GalNAcα-

Umbrella topology blocking agent (UTBA): As used herein, the term“umbrella topology blocking agent” refers to an agent which binds to anHA receptor having an umbrella topology glycan. In some embodiments, aUTBA binds to an HA receptor having an umbrella topology glycan found inhuman upper airways. A UBTA can bind to either an umbrella topologyglycan and/or to a cone topology glycan. In some embodiments, a UTBAselectively binds to an umbrella topology glycan with 50, 100, 150, or200% of its affinity for a cone topology glycan. In some embodiments aUTBA selectively binds to an umbrella topology glycan with 50-150% ofits affinity for a cone topology glycan. In some embodiments, a UTBAbinds to an umbrella topology glycan with about the same affinity as fora cone topology glycan. For example, in some embodiments, a UTBA bindsan umbrella topology glycan (e.g., 6′SLN-LN) with about 50-200%,50-150%, or about the same affinity to which it binds a cone topologyglycan (e.g., 3′SLN-LN). In some embodiments, a UTBA selectivelyinhibits the binding of an influenza virus particle (e.g., a human oravian influenza virus) to the HA receptor based on the glycan topologyof the receptor (e.g., umbrella or cone). In some embodiments, a UTBA isa polypeptide. In some such embodiments, a UTBA polypeptide has an aminoacid sequence that is substantially identical or substantiallyhomologous to that of a naturally-occurring polypeptide. In someembodiments, a UTBA polypeptide is an HA polypeptide. In someembodiments, a UTBA polypeptide is a naturally-occurring HA polypeptide,or a fragment thereof. In some embodiments, a UTBA polypeptide has anamino acid sequence that is not related to that of an HA polypeptide. Insome embodiments, a UTBA polypeptide is an antibody or fragment thereof.In some embodiments, a UTBA polypeptide is a lectin (e.g., SNA-1). Insome embodiments, a UTBA is not a polypeptide. In some embodiments, aUTBA is a small molecule. In some embodiments, a UTBA is a nucleic acid.

Umbrella topology glycan mimic: An “umbrella topology glycan mimic” isan agent, other than an umbrella topology glycan, that binds to bindingagents as described herein. In some embodiments, umbrella topologyglycan mimics are agents that bind to HA polypeptides. In some suchembodiments, umbrella topology glycan mimics are agents that interactwith HA polypeptide residues selected from the group consisting ofresidues 95, 98, 128, 130, 131, 132, 133, 135, 136, 137, 138, 145, 153,155, 156, 158, 159, 160, 183, 186, 187, 188, 189, 190, 192, 193, 194,195, 196, 219, 221, 222, 224, 225, 226, 227, 228, and combinationsthereof. In some such embodiments, umbrella topology glycan mimics areagents that interact with HA polypeptide residues selected from thegroup consisting of residues 130, 131, 132, 133, 135, 137, 155, 188,192, 193, 221, 226, 227, 228, and combinations thereof. In some suchembodiments, umbrella topology glycan mimics are agents that interactwith HA polypeptide residues selected from the group consisting ofresidues 160, 192, 193, and combinations thereof. Note that amino acidpositions stated above are based on H3 HA numbering. In someembodiments, an HA topology glycan mimic is an agent that competes withumbrella topology glycans for interaction with an HA polypeptide.

Umbrella topology specific blocking agent (UTSBA): As used herein, theterm “umbrella topology specific blocking agent” refers to an agentwhich binds to an HA receptor having an umbrella topology glycan foundin human upper airways. A UTSBA selectively binds an umbrella topologyglycan HA. For example, a UTSBA binds an umbrella topology glycan (e.g.,6′SLN-LN) with about at least 2, at least 4, at least 5, or at least 10times greater affinity than it binds to a cone topology glycan (e.g.,3′SLN-LN). Typically, the affinity of a UTSBA for an umbrella topologyglycan is greater than 1 nM. Typically the affinity of a UTSBA for acone topology glycan is less is at least within 2 to 3 orders ofmagnitude of the binding affinity of umbrella topology glycans to humanadapted HAs such as SC18, Mos99, Tx91, etc. and α2-6 binding plantlectins such as SNA-I. The binding affinity of UTSBA as measured by thedose-dependent direct binding assay (FIGS. 19 and 20) would typically beat least 1 nM. Typically the affinity of a UTSBA for a cone topologyglycan is at most 1 to 3 orders of magnitude less than the bindingaffinity of cone topology glycans to avian HAs such as Viet0405, Av18,etc. In some embodiments, a UTSBA selectively inhibits binding of aninfluenza virus particle (e.g., a human or avian influenza virus) to theHA receptor (e.g., an H1, H2 or H3 or a human-adapted H5, H7 or H9)based on glycan topology (e.g., umbrella or cone). In some embodiments,a UTSBA is a polypeptide. In some such embodiments, a UTSBA polypeptidehas an amino acid sequence that is that is substantially identical orsubstantially homologous to that of a naturally-occurring polypeptide.In some embodiments, a UTSBA polypeptide is an HA polypeptide. In someembodiments, a UTSBA polypeptide is a naturally-occurring HApolypeptide, or a fragment thereof. In some embodiments, a UTSBApolypeptide has an amino acid sequence that is not related to that of anHA polypeptide. In some embodiments, a UTSBA polypeptide is an antibodyor fragment thereof. In some embodiments, a UTSBA polypeptide is alectin (e.g., SNA-1). In some embodiments, a UTSBA is not a polypeptide.In some embodiments, a UTSBA is a small molecule. In some embodiments, aUTSBA is a nucleic acid.

Vaccination: As used herein, the term “vaccination” refers to theadministration of a composition intended to generate an immune response,for example to a disease-causing agent. For the purposes of the presentinvention, vaccination can be administered before, during, and/or afterexposure to a disease-causing agent, and in some embodiments, before,during, and/or shortly after exposure to the agent. In some embodiments,vaccination includes multiple administrations, appropriately spaced intime, of a vaccinating composition.

Variant: As used herein, the term “variant” is a relative term thatdescribes the relationship between a particular polypeptide (e.g., HApolypeptide) of interest and a “parent” polypeptide to which itssequence is being compared. A polypeptide of interest is considered tobe a “variant” of a parent polypeptide if the polypeptide of interesthas an amino acid sequence that is identical to that of the parent butfor a small number of sequence alterations at particular positions.Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% ofthe residues in the variant are substituted as compared with the parent.In some embodiments, a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1substituted residue as compared with a parent. Often, a variant has avery small number (e.g., fewer than 5, 4, 3, 2, or 1) number ofsubstituted functional residues (i.e., residues that participate in aparticular biological activity). Furthermore, a variant typically hasnot more than 5, 4, 3, 2, or 1 additions or deletions, and often has noadditions or deletions, as compared with the parent. Moreover, anyadditions or deletions are typically fewer than about 25, about 20,about 19, about 18, about 17, about 16, about 15, about 14, about 13,about 10, about 9, about 8, about 7, about 6, and commonly are fewerthan about 5, about 4, about 3, or about 2 residues. In someembodiments, the parent polypeptide is one found in nature. For example,a parent HA polypeptide may be one found in a natural (e.g., wild type)isolate of an influenza virus (e.g., a wild type HA).

Vector: As used herein, “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. In some embodiment, vectors are capable of extra-chromosomalreplication and/or expression of nucleic acids to which they are linkedin a host cell such as a eukaryotic or prokaryotic cell. Vectors capableof directing the expression of operatively linked genes are referred toherein as “expression vectors.”

Wild type: As is understood in the art, the phrase “wild type” generallyrefers to a normal form of a protein or nucleic acid, as is found innature. For example, wild type HA polypeptides are found in naturalisolates of influenza virus. A variety of different wild type HAsequences can be found in the NCBI influenza virus sequence database,available through the world wide web atncbi.nlm.nih.gov/genomes/FLU/FLU.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention provides binding agents (e.g., HA polypeptides, HApolypeptide variants, LSBAs, UTBAs, UTSBAs, etc.) that bind to umbrellatopology glycans. In some embodiments, the present invention providesbinding agents that bind to umbrella topology glycans found on HAreceptors of a particular target species. For example, in someembodiments, the present invention provides binding agents that bind toumbrella topology glycans found on human HA receptors, e.g., HAreceptors found on human epithelial cells, and particularly bindingagents that bind to umbrella topology glycans found on human HAreceptors in the upper respiratory tract.

The present invention provides binding agents that bind to HA receptorsfound on cells in the human upper respiratory tract, and in particularprovides binding agents that binds to such receptors (and/or to theirglycans, particularly to their umbrella glycans) with a designatedaffinity and/or specificity.

In some embodiments, binding agents in accordance with the presentinvention are or comprise HA polypeptide sequences. In some embodiments,binding agents in accordance with the present invention comprise an HApolypeptide sequence which differs from a parent naturally-occurring HApolypeptide sequence at one or more of residues selected from the groupconsisting of residues 130, 131, 132, 133, 135, 137, 155, 188, 192, 193,221, 226, 227, 228, and combinations thereof. In some embodiments,binding agents in accordance with the present invention comprise an HApolypeptide sequence which differs from a parent naturally-occurring HApolypeptide sequence at one or more of residues selected from the groupconsisting of residues 160, 192, 193, and combinations thereof.

The present invention encompasses the recognition that gaining anability to bind umbrella topology glycans (e.g., long α2-6 sialylatedglycans), and particularly an ability to bind with high affinity, mayconfer upon an HA polypeptide variant the ability to infect humans(where its parent HA polypeptide cannot). Without wishing to be bound byany particular theory, the present inventors propose that binding toumbrella topology glycans may be paramount, and in particular that lossof binding to other glycan types may not be required.

The present invention further provides various reagents and methodsassociated with binding agents in accordance with the invention (e.g.,HA polypeptides, HA polypeptide variants, UTBAs, UTSBAs, etc.)including, for example, systems for identifying them, strategies forpreparing them, antibodies that bind to them, and various diagnostic andtherapeutic methods relating to them. Further description of certainembodiments of these aspects, and others, of the present invention, ispresented below.

Hemagglutinin (HA)

Influenza viruses are RNA viruses which are characterized by a lipidmembrane envelope containing two glycoproteins, hemagglutinin (HA) andneuraminidase (NA), embedded in the membrane of the virus particular.There are 16 known HA subtypes and 9 NA subtypes, and differentinfluenza strains are named based on the number of the strain's HA andNA subtypes. Based on comparisons of amino acid sequence identity and ofcrystal structures, the HA subtypes have been divided into two maingroups and four smaller clades. The different HA subtypes do notnecessarily share strong amino acid sequence identity, but the overall3D structures of the different HA subtypes are similar to one another,with several subtle differences that can be used for classificationpurposes. For example, the particular orientation of the membrane-distalsubdomains in relation to a central a-helix is one structuralcharacteristic commonly used to determine HA subtype (Russell et al.,2004, Virology, 325:287; incorporated herein by reference).

HA exists in the membrane as a homotrimer of one of 16 subtypes, termedH1-H16. Only three of these subtypes (H1, H2, and H3) have thus farbecome adapted for human infection. One reported characteristic of HAsthat have adapted to infect humans (e.g., of HAs from the pandemic H1N1(1918) and H3N2 (1967-68) influenza subtypes) is their ability topreferentially bind to α2-6 sialylated glycans in comparison with theiravian progenitors that preferentially bind to α2-3 sialylated glycans(Skehel & Wiley, 2000, Annu Rev Biochem, 69:531; Rogers, & Paulson,1983, Virology, 127:361; Rogers et al., 1983, Nature, 304:76; Sauter etal., 1992, Biochemistry, 31:9609; Connor et al., 1994, Virology, 205:17;Tumpey et al., 2005, Science, 310:77; all of which are incorporatedherein by reference). The present invention, however, encompasses therecognition that ability to infect human hosts correlates less withbinding to glycans of a particular linkage, and more with binding toglycans of a particular topology. Thus, the present inventiondemonstrates that HAs that mediate infection of humans bind to umbrellatopology glycans, often showing preference for umbrella topology glycansover cone topology glycans (even though cone-topology glycans may beα2-6 sialylated glycans).

Several crystal structures of HAs from H1 (human and swine), H3 (avian)and H5 (avian) subtypes bound to sialylated oligosaccharides (of bothα2-3 and α2-6 linkages) are available and provide molecular insightsinto the specific amino acids that are involved in distinct interactionsof the HAs with these glycans (Eisen et al., 1997, Virology, 232:19; Haet al., 2001, Proc Natl Acad Sci USA, 98:11181; Ha et al., 2003,Virology, 309:209; Gamblin et al., 2004, Science, 303:1838; Stevens etal., 2004, Science, 303:1866; Russell et al., 2006, Glycoconj J 23:85;Stevens et al., 2006, Science, 312:404; all of which are incorporatedherein by reference).

For example, the crystal structures of H5 (A/duck/Singapore/3/97) aloneor bound to an α2-3 or an α2-6 sialylated oligosaccharide identifiescertain amino acids that interact directly with bound glycans, and alsoamino acids that are one or more degree of separation removed (Stevenset al., 2001, Proc Natl Acad Sci USA 98:11181; incorporated herein byreference). In some cases, conformation of these residues is differentin bound versus unbound states. For instance, Glu190, Lys193, and Gln226all participate in direct-binding interactions and have differentconformations in the bound versus the unbound state. The conformation ofAsn186, which is proximal to Glu190, is also significantly different inthe bound versus the unbound state.

Binding Agents

As noted above, the present invention encompasses the finding thatbinding to umbrella topology glycans correlates with ability to mediateinfection of particular hosts, including for example, humans.Accordingly, the present invention provides binding agents (e.g., HApolypeptides, HA polypeptide variants, LSBAs, UTBAs, UTSBAs, etc.) thatbind to umbrella glycans (and/or to umbrella topology glycan mimics). Insome embodiments, binding agents in accordance with the invention bindto umbrella glycans (and/or to umbrella topology glycan mimics) withhigh affinity. In some embodiments, binding agents in accordance withthe invention bind to a plurality of different umbrella topologyglycans, often with high affinity and/or specificity.

In some embodiments, binding agents in accordance with the inventionbind to umbrella topology glycans (e.g., long α2-6 sialylated glycanssuch as, for example, Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc-) withhigh affinity. For example, in some embodiments, binding agents inaccordance with the invention bind to umbrella topology glycans with anaffinity comparable to that observed for a wild type HA that mediatesinfection of a humans (e.g., H1N1 HA or H3N2 HA). In some embodiments,binding agents in accordance with the invention bind to umbrella glycanswith an affinity that is at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% of that observed under comparable conditions for awild type HA that mediates infection of humans. In some embodiments,binding agents in accordance with the invention bind to umbrella glycanswith an affinity that is greater than that observed under comparableconditions for a wild type HA that mediates infection of humans.

In some embodiments, binding affinity of binding agents in accordancewith the invention is assessed over a range of concentrations. Such astrategy provides significantly more information, particularly inmultivalent binding assays, than do single-concentration analyses. Insome embodiments, for example, binding affinities of binding agents inaccordance with the invention are assessed over concentrations rangingover at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10 or more fold.

In some embodiments, binding agents in accordance with the inventionshow high affinity if they show a saturating signal in a multivalentglycan array binding assay such as those described herein. In someembodiments, binding agents in accordance with the invention show highaffinity if they show a signal above about 400000 or more (e.g., aboveabout 500000, about 600000, about 700000, about 800000, etc.) in suchstudies. In some embodiments, binding agents as described herein showsaturating binding to umbrella glycans over a concentration range of atleast 2 fold, at least 3 fold, at least 4 fold, at least 5 fold or more,and in some embodiments over a concentration range as large as 10 foldor more.

Furthermore, in some embodiments, binding agents in accordance with theinvention bind to umbrella topology glycans (and/or to umbrella topologyglycan mimics) more strongly than they bind to cone topology glycans. Insome embodiments, binding agents in accordance with the invention show arelative affinity for umbrella glycans vs. cone glycans that is about10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, orabout 2.

In some embodiments, binding agents in accordance with the inventionbind to α2-6 sialylated glycans; in some embodiments, binding agents inaccordance with the invention bind preferentially to α2-6 sialylatedglycans. In some embodiments, binding agents in accordance with theinvention bind to a plurality of different α2-6 sialylated glycans. Insome embodiments, binding agents in accordance with the invention arenot able to bind to α2-3 sialylated glycans, and in some embodimentsbinding agents in accordance with the invention are able to bind to α2-3sialylated glycans.

In some embodiments, binding agents in accordance with the inventionbind to receptors found on human upper respiratory epithelial cells. Insome embodiments, binding agents in accordance with the invention bindto HA receptors in the bronchus and/or trachea. In some embodiments,binding agents in accordance with the invention are not able to bindreceptors in the deep lung, and in some embodiments, binding agents inaccordance with the invention are able to bind receptors in the deeplung.

In some embodiments, binding agents in accordance with the inventionbind to at least about 10%, about 15%, about 20%, about 25%, about 30%about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,or more of the glycans found on HA receptors in human upper respiratorytract tissues (e.g., epithelial cells).

In some embodiments, binding agents in accordance with the inventionbind to one or more of the glycans illustrated in FIGS. 9A-B. In someembodiments, binding agents in accordance with the invention bind tomultiple glycans illustrated in FIGS. 9A-B. In some embodiments, bindingagents in accordance with the invention bind with high affinity and/orspecificity to glycans illustrated in FIGS. 9A-B. In some embodiments,binding agents in accordance with the invention bind to glycansillustrated in FIGS. 9A-B preferentially as compared with their bindingto glycans illustrated in FIG. 8. In some embodiments, binding agents inaccordance with the invention bind to an oligosaccharide of thefollowing form:

Neu5Acα2-6Sug1-Sug2-Sug3

where:

-   -   1. Neu5Ac α2-6 is always or almost always at the non-reducing        end;    -   2. Sug1:        -   a. is a hexose (frequently Gal or Glc) or hexosamine (GlcNAc            or GalNAc) in α or β configuration (frequently β- for N- and            O-linked extension and α- in the case of GalNAcα- that is            O-linked to glycoprotein);        -   b. no sugars other than Neu5Acα2-6 should be attached to any            of the non-reducing positions of Sug1 (except when Sug1 is            GalNAcα- that is O-linked to the glycoprotein); and/or        -   c. non-sugar moieties such as sulfate, phosphate, guanidium,            amine, N-acetyl, etc. can be attached to non-reducing            positions (typically 6 position) of Sug1 to improve contacts            with HA;    -   3. Sug2 and/or Sug3:        -   a. hexose (frequently Gal or Glc) or hexosamine (GlcNAc or            GalNAc) in α or β configuration (frequently β); and/or        -   b. sugars (such as Fuc) or non-sugar moieties such as            sulfate, phosphate, guanidium, amine, N-acetyl, etc. can be            attached to non-reducing positions of Sug2, Sug3, and/or            Sug4;    -   4. Linkage between any two sugars in the oligosaccharide apart        from Neu5Acα2-6 linkage can be 1-2, 1-3, 1-4, and/or 1-6        (typically 1-3 or 1-4); and/or    -   5. Structure where Neu5Acα2-6 is linked GalNAcα that is O-linked        to the glycoprotein and additional sugars are linked to the        non-reducing end of GalNAcα for example        -   i. Neu5Acα2-6(Neu5Acα2-3Galβ1-3)GalNAcα-        -   ii. Neu5Acα2-6(Galβ1-3)GalNAcα-

The present invention provides binding agents with designated bindingspecificity, and also provides binding agents with designated bindingcharacteristics with respect to umbrella glycans.

Certain particular binding agents provided by the present invention aredescribed in more detail below.

HA Polypeptides

In some embodiments, binding agents in accordance with the invention areHA polypeptides. For example, the present invention provides isolated HApolypeptides with designated binding specificity, and also providesengineered HA polypeptides with designated binding characteristics withrespect to umbrella glycans.

In some embodiments, provided HA polypeptides with designated bindingcharacteristics are H1 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H2 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H3 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H4 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H5 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H6 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H7 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H8 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H9 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H10 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H11 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H12 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H13 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H14 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H15 polypeptides. In some embodiments, HApolypeptides in accordance with the invention with designated bindingcharacteristics are H16 polypeptides.

In some embodiments, HA polypeptides in accordance with the invention donot include the H1 protein from any of the strains: A/SouthCarolina/1/1918; A/Puerto Rico/8/1934; A/Taiwan/1/1986; A/Texas/36/1991;A/Beijing/262/1995; A/Johannesburg/92/1996; A/New Caledonia/20/1999;A/Solomon Islands/3/2006.

In some embodiments, HA polypeptides in accordance with the inventionare not the H2 protein from any of the strains of the Asian flu epidemicof 1957-58). In some embodiments, HA polypeptides in accordance with theinvention do not include the H2 protein from any of the strains:A/Japan/305+/1957; A/Singapore/1/1957; A/Taiwan/1/1964; A/Taiwan/1/1967.

In some embodiments, HA polypeptides in accordance with the invention donot include the H3 protein from any of the strains: A/Aichi/2/1968;A/Philippines/2/1982; A/Mississippi/1/1985; A/Leningrad/360/1986;A/Sichuan/2/1987; A/Shanghai/11/1987; A/Beijing/353/1989;A/Shandong/9/1993; A/Johannesburg/33/1994; A/Nanchang/813/1995;A/Sydney/5/1997; A/Moscow/10/1999; A/Panama/2007/1999; A/Wyoming/3/2003;A/Oklahoma/323/2003; A/California/7/2004; A/Wisconsin/65/2005.

Variant HA Polypeptides

In some embodiments, a provided HA polypeptide is a variant of a parentHA polypeptide in that its amino acid sequence is identical to that ofthe parent HA but for a small number of particular sequence alterations.In some embodiments, the parent HA is an HA polypeptide found in anatural isolate of an influenza virus (e.g., a wild type HApolypeptide).

In some embodiments, HA polypeptide variants in accordance with theinvention have different glycan binding characteristics than theircorresponding parent HA polypeptides. In some embodiments, HA variantpolypeptides in accordance with the invention have greater affinityand/or specificity for umbrella glycans (e.g., as compared with for coneglycans) than do their cognate parent HA polypeptides. In someembodiments, such HA polypeptide variants are engineered variants.

In some embodiments, HA variant polypeptides in accordance with theinvention have greater affinity and/or specificity for umbrella glycansas compared with their cognate parent HA polypeptides. In someembodiments, HA variant polypeptides in accordance with the inventionhave reduced affinity and/or specificity for cone topology glycans ascompared with their cognate parent HA polypeptides. In some embodiments,HA variant polypeptides in accordance with the invention have greateraffinity and/or specificity for umbrella glycans and reduced affinityand/or specificity for cone topology glycans as compared with theircognate parent HA polypeptides.

In some embodiments, HA variant polypeptides in accordance with theinvention have greater affinity and/or specificity for α2-6 glycans ascompared with their cognate parent HA polypeptides. In some embodiments,HA variant polypeptides in accordance with the invention have reducedaffinity and/or specificity for α2-3 glycans as compared with theircognate parent HA polypeptides. In some embodiments, HA variantpolypeptides in accordance with the invention have greater affinityand/or specificity for α2-6 and α2-3 glycans as compared with theircognate parent HA polypeptides. In some embodiments, HA variantpolypeptides in accordance with the invention have greater affinityand/or specificity for α2-6 glycans and reduced affinity and/orspecificity for α2-3 glycans as compared with their cognate parent HApolypeptides.

In some embodiments, HA variant polypeptides in accordance with theinvention have greater affinity and/or specificity for umbrella topologyglycans and α2-3 glycans as compared with their cognate parent HApolypeptides. In some embodiments, HA variant polypeptides in accordancewith the invention have greater affinity and/or specificity for umbrellatopology glycans and reduced affinity and/or specificity for α2-3glycans as compared with their cognate parent HA polypeptides.

In some embodiments, HA variant polypeptides in accordance with theinvention have greater affinity and/or specificity for α2-6 glycans andcone topology glycans as compared with their cognate parent HApolypeptides. In some embodiments, HA variant polypeptides in accordancewith the invention have greater affinity and/or specificity for α2-6glycans and reduced affinity and/or specificity for cone topologyglycans as compared with their cognate parent HA polypeptides.

In some embodiments, HA polypeptide variants in accordance with theinvention contain one or more sequence alterations that are consistentwith HA sequences found in a different HA subtype. To give but oneparticular example, in some embodiments, an H5 HA polypeptide variant inaccordance with the invention contains one or more sequence alterationswhich make the H5 HA polypeptide variant more closely resemble an H2 HApolypeptide. To give another particular example, in some embodiments, anH5 HA polypeptide variant in accordance with the invention contains oneor more sequence alterations which make the H5 HA polypeptide variantmore closely resemble an H1 HA polypeptide.

The present invention particularly encompasses the recognition that HApolypeptide variants (e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10,H11, H12, H13, H14, H15, or H16 HA polypeptide variants) with alteredglycosylation can show 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold,50-fold, 100-fold, 1000-fold, 10,000-fold, or greater affinity to humanHA receptors as compared with a reference HA polypeptide. The presentinvention also particularly encompasses the recognition that HApolypeptide variants (e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10,H11, H12, H13, H14, H15, or H16 HA polypeptide variants) withalterations in the HA loop region, can show 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold, 10,000-fold, orgreater affinity to human HA receptors as compared with a reference HApolypeptide (e.g., an HA polypeptide of any of SEQ ID NOs: 43-55).

The present invention particularly encompasses the recognition that HApolypeptide variants (e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10,H11, H12, H13, H14, H15, or H16 HA polypeptide variants) with alteredglycosylation can show 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold,50-fold, 100-fold, 1000-fold, 10,000-fold, or greater specificity forhuman HA receptors as compared with a reference HA polypeptide. Thepresent invention also particularly encompasses the recognition that HApolypeptide variants (e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10,H11, H12, H13, H14, H15, or H16 HA polypeptide variants) withalterations in the HA loop region, can show 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold, 10,000-fold, orgreater specificity for human HA receptors as compared with a referenceHA polypeptide (e.g., an HA polypeptide of any of SEQ ID NOs: 43-55).

In some embodiments, the reference HA polypeptide is the HA from A/SouthCarolina/1/18 (H1N1) (SEQ ID NO: 43). In some embodiments, the referenceHA polypeptide is the HA from A/Brisbane/59/07 (SEQ ID NO: 44). In someembodiments, the reference HA polypeptide is the HA fromA/California/04/09 (SEQ ID NO: 45). In some embodiments, the referenceHA polypeptide is the HA from A/Albany/6/58 (H2N2) (SEQ ID NO: 46). Insome embodiments, the reference HA polypeptide is the HA fromA/Aichi/1/68 (SEQ ID NO: 47). In some embodiments, the reference HApolypeptide is the HA from A/Moscow/10/99 (SEQ ID NO: 48). In someembodiments, the reference HA polypeptide is the HA from A/Perth/16/09(SEQ ID NO: 49). In some embodiments, the reference HA polypeptide isthe HA from A/Vietnam/1203/04 (SEQ ID NO: 50). In some embodiments, thereference HA polypeptide is the HA from A/Egypt/2786-NAMRU3/06 (SEQ IDNO: 51). In some embodiments, the reference HA polypeptide is the HAfrom A/New York/107/03 (SEQ ID NO: 52). In some embodiments, thereference HA polypeptide is the HA from A/Hongkong/486/97 (SEQ ID NO:53). In some embodiments, the reference HA polypeptide is the HA fromA/Hongkong/213/03 (SEQ ID NO: 54). In some embodiments, the reference HApolypeptide is the HA from A/Indonesia/5/05 (SEQ ID NO: 55).

In some embodiments, HA polypeptide variants with altered glycan bindingcharacteristics have one or more sequence alternations in residueswithin or affecting the glycan binding site. In some embodiments, suchsubstitutions are of amino acids that interact directly with boundglycan; in some embodiments, such substitutions are of amino acids thatare one degree of separation removed from those that interact with boundglycan, in that the one degree of separation removed—amino acids either(1) interact with the direct-binding amino acids; (2) otherwise affectthe ability of the direct-binding amino acids to interact with glycan,but do not interact directly with glycan themselves; or (3) otherwiseaffect the ability of the direct-binding amino acids to interact withglycan, and also interact directly with glycan themselves. HApolypeptide variants in accordance with the invention containsubstitutions of one or more direct-binding amino acids, one or morefirst degree of separation—amino acids, one or more second degree ofseparation—amino acids, or any combination of these. In someembodiments, HA polypeptide variants in accordance with the inventionmay contain substitutions of one or more amino acids with even higherdegrees of separation.

In some embodiments, HA polypeptide variants with altered glycan bindingcharacteristics have sequence alterations in residues that make contactwith sugars beyond Neu5Ac and Gal (see, for example, FIG. 7).

In some embodiments, HA polypeptide variants have at least one aminoacid substitution, as compared with a wild type parent HA. In someembodiments, HA polypeptide variants in accordance with the inventionhave at least two, three, four, five or more amino acid substitutions ascompared with a cognate wild type parent HA; in some embodiments HApolypeptide variants in accordance with the invention have two, three,or four amino acid substitutions. In some embodiments, all such aminoacid substitutions are located within the glycan binding site.

In some embodiments, HA polypeptide variants in accordance with theinvention contain one or more amino acid substitutions as described inany of U.S. Patent Publication Number 2009/0269342 and 2010/0004195, andin U.S. patent application Ser. No. 12/829931, filed Jul. 2, 2010,entitled “COMPOSITIONS AND METHODS FOR DIAGNOSING AND/OR TREATINGINFLUENZA INFECTION” (all of which are incorporated herein byreference).

In some embodiments, HA polypeptide variants have sequence substitutionsat positions corresponding to one or more of residues 95, 98, 128, 130,131, 132, 133, 135, 136, 137, 138, 145, 153, 155, 156, 158, 159, 160,183, 186, 187, 188, 189, 190, 192, 193, 194, 195, 196, 219, 221, 222,224, 225, 226, 227, and 228. In some embodiments, HA polypeptidevariants, particularly H5 polypeptide variants, have one or more aminoacid substitutions relative to a wild type parent HA (e.g., H5) atresidues selected from the group consisting of residues 95, 98, 128,130, 131, 132, 133, 135, 136, 137, 138, 145, 153, 155, 156, 158, 159,160, 183, 186, 187, 188, 189, 190, 192, 193, 194, 195, 196, 219, 221,222, 224, 225, 226, 227, and 228. In some embodiments, HA polypeptidevariants, particularly H5 polypeptide variants, have one or more aminoacid substitutions relative to a wild type parent HA (e.g., H5) at any1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37residues selected from the group consisting of residues 95, 98, 128,130, 131, 132, 133, 135, 136, 137, 138, 145, 153, 155, 156, 158, 159,160, 183, 186, 187, 188, 189, 190, 192, 193, 194, 195, 196, 219, 221,222, 224, 225, 226, 227, and 228.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions that reduce or abolishglycosylation at a site corresponding to amino acid position 158. Insome embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions that affect and/or alter theidentity and/or structure of the glycan linked to a site correspondingto amino acid position 158. In some embodiments, such a sequencesubstitution is a mutation at a site corresponding to position 158,e.g., Asn158Xaa, wherein Xaa is any amino acid other than Asn. In someembodiments, such a sequence substitution is a mutation at a sitecorresponding to position 160, e.g., Thr160Xaa, wherein Xaa is any aminoacid other than Asn. In some embodiments, such a sequence substitutioncomprises the mutation Thr160Ala. In some embodiments, a sequencesubstitution that reduces, abolishes, affects, or alters glycosylationat a site corresponding to amino acid position 158 can make a non-H2 HApolypeptide (e.g., an H5 HA polypeptide) more closely resemble (e.g.,both structurally and functionally) an H2 HA polypeptide. In someembodiments, a mutation at a site corresponding to position 160 (e.g.,Thr160Xaa, such as Thr160Ala) can make a non-H2 HA polypeptide (e.g., anH5 HA polypeptide) more closely resemble (e.g., both structurally andfunctionally) an H2 HA polypeptide.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to one or more of residues 226,228, and 160. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to residues 226,228, and 160. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to residues 226and 160. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to residues 228and 160. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to residues 226and 228.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to one or more of residues 226,228, and 158. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to residues 226,228, and 158. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to residues 226and 158. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to residues 228and 158. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to residues 226and 228.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions that include a deletion in one ormore of the loop regions of an HA polypeptide. In some embodiments, HApolypeptide variants (e.g., H5 HA polypeptide variants) have sequencesubstitutions that include a deletion at a site corresponding to the128-137 loop region of an HA polypeptide. In some embodiments, HApolypeptide variants (e.g., H5 HA polypeptide variants) have sequencesubstitutions that include a deletion at one or more of amino acidpositions corresponding to residues 128, 129, 130, 131, 132, 133, 134,135, 136, and/or 137 of an HA polypeptide. In some embodiments, HApolypeptide variants (e.g., H5 HA polypeptide variants) have sequencesubstitutions that include a deletion at a site corresponding to the128-134 loop region of an HA polypeptide. In some embodiments, HApolypeptide variants (e.g., H5 HA polypeptide variants) have sequencesubstitutions that include a deletion at one or more of amino acidpositions corresponding to residues 128, 129, 130, 131, 132, 133, and/or134 of an HA polypeptide. In some embodiments, HA polypeptide variants(e.g., H5 HA polypeptide variants) have sequence substitutions thatinclude a deletion of an amino acid corresponding to residue 130. Insome embodiments, such loop region substitutions can make a non-H2 HApolypeptide (e.g., an H5 HA polypeptide) more closely resemble (e.g.,both structurally and functionally) an H2 HA polypeptide. In someembodiments, a deletion of an amino acid corresponding to residue 130can make a non-H2 HA polypeptide (e.g., an H5 HA polypeptide) moreclosely resemble (e.g., both structurally and functionally) an H2 HApolypeptide.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to one or more of residues 131,132, 133, 135, 137, 155, 188, 192, 193, 221, 226, 227, 228, and 130. Insome embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to any 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, or 14 of residues 131, 132, 133, 135, 137, 155, 188,192, 193, 221, 226, 227, 228, and 130. In some embodiments, HApolypeptide variants (e.g., H5 HA polypeptide variants) have sequencesubstitutions relative to a wild type parent HA (e.g., H5 HA) atpositions corresponding to (1) 130, and (2) one or more of residues 131,132, 133, 135, 137, 155, 188, 192, 193, 221, 226, 227, and 228. In someembodiments, HA polypeptide variants (e.g., H5 HA polypeptide variants)have sequence substitutions relative to a wild type parent HA (e.g., H5HA) at positions corresponding to (1) 130, and (2) any 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, or 13 of residues 131, 132, 133, 135, 137, 155,188, 192, 193, 221, 226, 227, and 228.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to one or more of residues 131,132, 135, 188, 192, 221, and 130. In some embodiments, HA polypeptidevariants (e.g., H5 HA polypeptide variants) have sequence substitutionsrelative to a wild type parent HA (e.g., H5 HA) at positionscorresponding to any 1, 2, 3, 4, 5, 6, or 7 of residues 131, 132, 135,188, 192, 221, and 130. In some embodiments, HA polypeptide variants(e.g., H5 HA polypeptide variants) have sequence substitutions relativeto a wild type parent HA (e.g., H5 HA) at positions corresponding to (1)130, and (2) one or more of residues 131, 132, 135, 188, 192, and 221.In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to (1) 130, and (2) any 1, 2,3, 4, 5, or 6 of residues 131, 132, 135, 188, 192, and 221.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to one or more of residues 133,137, 155, 193, 226, 227, 228, and 130. In some embodiments, HApolypeptide variants (e.g., H5 HA polypeptide variants) have sequencesubstitutions relative to a wild type parent HA (e.g., H5 HA) atpositions corresponding to any 1, 2, 3, 4, 5, 6, 7, or of residues 133,137, 155, 193, 226, 227, 228, and 130. In some embodiments, HApolypeptide variants (e.g., H5 HA polypeptide variants) have sequencesubstitutions relative to a wild type parent HA (e.g., H5 HA) atpositions corresponding to (1) 130, and (2) one or more of residues 133,137, 155, 193, 226, 227, and 228. In some embodiments, HA polypeptidevariants (e.g., H5 HA polypeptide variants) have sequence substitutionsrelative to a wild type parent HA (e.g., H5 HA) at positionscorresponding to (1) 130, and (2) any 1, 2, 3, 4, 5, 6, or 7 of residues133, 137, 155, 193, 226, 227, and 228.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to one or more of residues 130,192, and 193. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to any 1, 2, or3 of residues 130, 192, 193. In some embodiments, HA polypeptidevariants (e.g., H5 HA polypeptide variants) have sequence substitutionsrelative to a wild type parent HA (e.g., H5 HA) at positionscorresponding to (1) 130, and (2) one or both of residues 192 and 193.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to one or more of residues 131,132, 133, 135, 137, 155, 158, 160, 188, 192, 193, 221, 226, 227, 228,and 130. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to any 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of residues 131, 132,133, 135, 137, 155, 158, 160, 188, 192, 193, 221, 226, 227, 228, and130. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to (1) 130, and(2) one or more of residues 131, 132, 133, 135, 137, 155, 158, 160, 188,192, 193, 221, 226, 227, and 228. In some embodiments, HA polypeptidevariants (e.g., H5 HA polypeptide variants) have sequence substitutionsrelative to a wild type parent HA (e.g., H5 HA) at positionscorresponding to (1) 130, and (2) any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, or 15 of residues 131, 132, 133, 135, 137, 155, 158, 160,188, 192, 193, 221, 226, 227, and 228.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to one or more of residues 137,188, 192, 193, 226, 228, and 130. In some embodiments, HA polypeptidevariants (e.g., H5 HA polypeptide variants) have sequence substitutionsrelative to a wild type parent HA (e.g., H5 HA) at positionscorresponding to any 1, 2, 3, 4, 5, 6, or 7 of residues 137, 188, 192,193, 226, 228, and 130. In some embodiments, HA polypeptide variants(e.g., H5 HA polypeptide variants) have sequence substitutions relativeto a wild type parent HA (e.g., H5 HA) at positions corresponding to (1)130, and (2) one or more of residues 137, 188, 192, 193, 226, and 228.In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to (1) 130, and (2) any 1, 2,3, 4, 5, or 6 of residues 137, 188, 192, 193, 226, and 228.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to one or more of residues 137,188, 192, 193, 226, 227, 228, 131, 132, 133, and 130. In someembodiments, HA polypeptide variants (e.g., H5 HA polypeptide variants)have sequence substitutions relative to a wild type parent HA (e.g., H5HA) at positions corresponding to any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or11 of residues 137, 188, 192, 193, 226, 227, 228, 131, 132, 133, and130. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to (1) 130, and(2) one or more of residues 137, 188, 192, 193, 226, 227, 228, 131, 132,and 133. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to (1) 130, and(2) any 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of residues 137, 188, 192, 193,226, 227, 228, 131, 132, and 133.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to one or more of residues 227,131, 132, 133, and 130. In some embodiments, HA polypeptide variants(e.g., H5 HA polypeptide variants) have sequence substitutions relativeto a wild type parent HA (e.g., H5 HA) at positions corresponding to any1, 2, 3, 4, or 5 of residues 227, 131, 132, 133, and 130. In someembodiments, HA polypeptide variants (e.g., H5 HA polypeptide variants)have sequence substitutions relative to a wild type parent HA (e.g., H5HA) at positions corresponding to (1) 130, and (2) one or more ofresidues 227, 131, 132, and 133. In some embodiments, HA polypeptidevariants (e.g., H5 HA polypeptide variants) have sequence substitutionsrelative to a wild type parent HA (e.g., H5 HA) at positionscorresponding to (1) 130, and (2) any 1, 2, 3, or 4 of residues 227,131, 132, and 133.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to one or more of residues 131,133, 137, 155, 188, 192, 193, 226, 227, 228, and 130. In someembodiments, HA polypeptide variants (e.g., H5 HA polypeptide variants)have sequence substitutions relative to a wild type parent HA (e.g., H5HA) at positions corresponding to any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or11 of residues 131, 133, 137, 155, 188, 192, 193, 226, 227, 228, and130. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to (1) 130, and(2) one or more of residues 131, 133, 137, 155, 188, 192, 193, 226, 227,and 228. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to (1) 130, and(2) any 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of residues 131, 133, 137, 155,188, 192, 193, 226, 227, and 228.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to one or more of residues 131,133, 137, 155, 188, 192, 193, 226, 228, and 130. In some embodiments, HApolypeptide variants (e.g., H5 HA polypeptide variants) have sequencesubstitutions relative to a wild type parent HA (e.g., H5 HA) atpositions corresponding to any 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ofresidues 131, 133, 137, 155, 188, 192, 193, 226, 228, and 130. In someembodiments, HA polypeptide variants (e.g., H5 HA polypeptide variants)have sequence substitutions relative to a wild type parent HA (e.g., H5HA) at positions corresponding to (1) 130, and (2) one or more ofresidues 131, 133, 137, 155, 188, 192, 193, 226, and 228. In someembodiments, HA polypeptide variants (e.g., H5 HA polypeptide variants)have sequence substitutions relative to a wild type parent HA (e.g., H5HA) at positions corresponding to (1) 130, and (2) any 1, 2, 3, 4, 5, 6,7, 8, or 9 of residues 131, 133, 137, 155, 188, 192, 193, 226, and 228.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to one or more of residues 131,133, 137, 155, 159, 160, 188, 192, 193, 226, 227, 228, and 130. In someembodiments, HA polypeptide variants (e.g., H5 HA polypeptide variants)have sequence substitutions relative to a wild type parent HA (e.g., H5HA) at positions corresponding to any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, or 13 of residues 131, 133, 137, 155, 159, 160, 188, 192, 193, 226,227, 228, and 130. In some embodiments, HA polypeptide variants (e.g.,H5 HA polypeptide variants) have sequence substitutions relative to awild type parent HA (e.g., H5 HA) at positions corresponding to (1) 130,and (2) one or more of residues 131, 133, 137, 155, 159, 160, 188, 192,193, 226, 227, and 228. In some embodiments, HA polypeptide variants(e.g., H5 HA polypeptide variants) have sequence substitutions relativeto a wild type parent HA (e.g., H5 HA) at positions corresponding to (1)130, and (2) any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of residues131, 133, 137, 155, 159, 160, 188, 192, 193, 226, 227, and 228.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to one or more of residues 131,133, 137, 155, 159, 160, 188, 192, 193, 226, 228, and 130. In someembodiments, HA polypeptide variants (e.g., H5 HA polypeptide variants)have sequence substitutions relative to a wild type parent HA (e.g., H5HA) at positions corresponding to any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or 12 of residues 131, 133, 137, 155, 159, 160, 188, 192, 193, 226, 228,and 130. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to (1) 130, and(2) one or more of residues 131, 133, 137, 155, 159, 160, 188, 192, 193,226, and 228. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to (1) 130, and(2) any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of residues 131, 133, 137,155, 159, 160, 188, 192, 193, 226, and 228.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to one or more of residues 137,188, 192, 193, 226, 228, 131, 132, 133, 221, 227, and 130. In someembodiments, HA polypeptide variants (e.g., H5 HA polypeptide variants)have sequence substitutions relative to a wild type parent HA (e.g., H5HA) at positions corresponding to any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or 12 of residues 137, 188, 192, 193, 226, 228, 131, 132, 133, 221, 227,and 130. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to (1) 130, and(2) one or more of residues 137, 188, 192, 193, 226, 228, 131, 132, 133,221, and 227. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to (1) 130, and(2) any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of residues 137, 188, 192,193, 226, 228, 131, 132, 133, 221, and 227.

In some embodiments, HA polypeptide variants (e.g., H5 HA polypeptidevariants) have sequence substitutions relative to a wild type parent HA(e.g., H5 HA) at positions corresponding to one or more of residues 131,132, 133, 221, 227, and 130. In some embodiments, HA polypeptidevariants (e.g., H5 HA polypeptide variants) have sequence substitutionsrelative to a wild type parent HA (e.g., H5 HA) at positionscorresponding to any 1, 2, 3, 4, 5, or 6 of residues 131, 132, 133, 221,227, and 130. In some embodiments, HA polypeptide variants (e.g., H5 HApolypeptide variants) have sequence substitutions relative to a wildtype parent HA (e.g., H5 HA) at positions corresponding to (1) 130, and(2) one or more of residues 131, 132, 133, 221, and 227. In someembodiments, HA polypeptide variants (e.g., H5 HA polypeptide variants)have sequence substitutions relative to a wild type parent HA (e.g., H5HA) at positions corresponding to (1) 130, and (2) any 1, 2, 3, 4, or 5of residues 131, 132, 133, 221, and 227.

In some embodiments, an HA polypeptide variant, and particularly an H5polypeptide variant, has one or more amino acid substitutions relativeto a wild type parent HA at residues selected from amino acids locatedin the region of the receptor that directly binds to the glycan,including but not limited to residues 98, 136, 153, 155, 183, and 194.In some embodiments, an HA polypeptide variant in accordance with theinvention, and particularly an H5 polypeptide variant, has one or moreamino acid substitutions relative to a wild type parent HA at residuesselected from amino acids located adjacent to the region of the receptorthat directly binds the glycan, including but not limited to (a)residues 98 and 195, (b) residues 98, 138, 186, 187, 195, and 228), or(c) residues 138, 186, 187, and 228.

In some embodiments, an HA polypeptide variant in accordance with theinvention, and particularly an H5 variant has one or more of thefollowing amino acid substitutions: Ser132Thr, Ala133Thr, Ser133Thr,Ser137Ala, Ser137Arg, Ile155Thr, Lys156Glu, Asn158Xaa (wherein Xaa=anyamino acid besides Asn), Thr160Ala, Asn186Pro, Asp187Ser, Asp187Thr,Ala188Glu, Ala188Asp, Ala189Gln, Ala189Lys, Ala189Thr, Glu190Asp,Glu190Thr, Thr192Arg/Lys, Lys193Arg, Lys193Asn, Lys193His, Lys193Ser,Lys/Arg193Thr/Ala/Met/Val, Ser221Pro, Gly225Asp, Gln226Ile, Gln226Leu,Gln226Val, Ser227Ala, Gly228Ser.

In some embodiments, an HA polypeptide variant (e.g., an H5 HApolypeptide variant) in accordance with the invention has an amino acidsubstitution at a position corresponding to residue 192, which switchesthe charge at that position. In some embodiments, an HA polypeptidevariant (e.g., an H5 HA polypeptide variant) in accordance with theinvention has an amino acid substitution at a position corresponding toresidue 193, which switches the charge at that position. For example, insome embodiments, an HA polypeptide (e.g., an H5 HA polypeptide) inaccordance with the invention has a Thr or a hydrophobic residue (e.g.,Val or Ile) at a position corresponding to residue 192, and an HApolypeptide variant (e.g., an H5 HA polypeptide variant) (e.g., ahuman-adapted variant) has a hydrophilic residue at a positioncorresponding to residue 192. In some embodiments, an HA polypeptidevariant (e.g., an H5 HA polypeptide variant) (e.g., a human-adaptedvariant) has a hydrophilic residue at a position corresponding toresidue 192. To give another example, in some embodiments, an HApolypeptide (e.g., an H5 HA polypeptide) in accordance with theinvention has a Thr or a hydrophobic residue (e.g., Val or Ile) at aposition corresponding to residue 192, and an HA polypeptide variant(e.g., an H5 HA polypeptide variant) (e.g., a human-adapted variant) hasa basic residue (e.g., Lys or Arg) at a position corresponding toresidue 192. In some embodiments, an HA polypeptide variant (e.g., an H5HA polypeptide variant) (e.g., a human-adapted variant) has a basicresidue (e.g., Lys or Arg) at a position corresponding to residue 192.To give yet another example, in some embodiments, an HA polypeptide(e.g., an H5 HA polypeptide) in accordance with the invention has abasic residue (e.g., Lys or Arg) at a position corresponding to residue193, and an HA polypeptide variant (e.g., an H5 HA polypeptide variant)(e.g., a human-adapted variant) has a neutral or acidic residue at aposition corresponding to residue 193. In some embodiments, an HApolypeptide variant (e.g., an H5 HA polypeptide variant) (e.g., ahuman-adapted variant) has a neutral or acidic residue at a positioncorresponding to residue 193. In some embodiments, an HA polypeptidevariant (e.g., an H5 HA polypeptide variant) (e.g., a human-adaptedvariant) has a Thr, Ala, Met, or Val at a position corresponding toresidue 193.

In some embodiments, human adaptation of HA polypeptides (e.g., H5 HApolypeptides) is associated with the propert(ies) of the residue atposition 188. In H5 HA, residue 188 is frequently Ala, which makescontacts with Thr or a hydrophobic residue at 192. In contrast, in H2HA, residue 188 is frequently Glu or Asp, which makes contacts with Argor Lys at 192. Hence, in some embodiments, an HA polypeptide variant(e.g., an H5 HA polypeptide variant) has a Glu at position 188. In someembodiments, an HA polypeptide variant (e.g., an H5 HA polypeptidevariant) has an Asp at position 188. In some embodiments, an HApolypeptide variant (e.g., an H5 HA polypeptide variant) has anAla188Glu substitution. In some embodiments, an HA polypeptide variant(e.g., an H5 HA polypeptide variant) has an Ala188Asp substitution.

In some embodiments, an HA polypeptide (e.g., an H5 HA polypeptide) hasan Ala at a position corresponding to residue 188 and a Thr at aposition corresponding to residue 192. In some embodiments, an HApolypeptide (e.g., an H5 HA polypeptide) has an Ala at a positioncorresponding to residue 188 and a hydrophobic residue at a positioncorresponding to residue 192. In some embodiments, an HA polypeptidevariant (e.g., an H5 HA polypeptide variant) has a Glu at a positioncorresponding to residue 188 and an Arg at a position corresponding toresidue 192. In some embodiments, an HA polypeptide variant (e.g., an H5HA polypeptide variant) has an Asp at a position corresponding toresidue 188 and an Arg at a position corresponding to residue 192. Insome embodiments, an HA polypeptide variant (e.g., an H5 HA polypeptidevariant) has a Glu at a position corresponding to residue 188 and a Lysat a position corresponding to residue 192. In some embodiments, an HApolypeptide variant (e.g., an H5 HA polypeptide variant) has an Asp at aposition corresponding to residue 188 and a Lys at a positioncorresponding to residue 192.

In some embodiments, an HA polypeptide (e.g., an H5 HA polypeptide) hasan Ala at a position corresponding to residue 188, a Thr at a positioncorresponding to residue 192, and a Lys at a position corresponding toresidue 193. In some embodiments, an HA polypeptide (e.g., an H5 HApolypeptide) has an Ala at a position corresponding to residue 188, ahydrophobic residue at a position corresponding to residue 192, and aLys at a position corresponding to residue 193. In some embodiments, anHA polypeptide (e.g., an H5 HA polypeptide) has an Ala at a positioncorresponding to residue 188, a Thr at a position corresponding toresidue 192, and an Arg at a position corresponding to residue 193. Insome embodiments, an HA polypeptide (e.g., an H5 HA polypeptide) has anAla at a position corresponding to residue 188, a hydrophobic residue ata position corresponding to residue 192, and an Arg at a positioncorresponding to residue 193. In some embodiments, an HA polypeptidevariant (e.g., an H5 HA polypeptide variant) has a Glu at a positioncorresponding to residue 188, an Arg at a position corresponding toresidue 192, and a Thr at a position corresponding to residue 193. Insome embodiments, an HA polypeptide variant (e.g., an H5 HA polypeptidevariant) has an Asp at a position corresponding to residue 188, an Argat a position corresponding to residue 192, and a Thr at a positioncorresponding to residue 193. In some embodiments, an HA polypeptidevariant (e.g., an H5 HA polypeptide variant) has a Glu at a positioncorresponding to residue 188, a Lys at a position corresponding toresidue 192, and a Thr at a position corresponding to residue 193. Insome embodiments, an HA polypeptide variant (e.g., an H5 HA polypeptidevariant) has an Asp at a position corresponding to residue 188, a Lys ata position corresponding to residue 192, and a Thr at a positioncorresponding to residue 193. In some embodiments, an HA polypeptidevariant (e.g., an H5 HA polypeptide variant) has a Glu at a positioncorresponding to residue 188, an Arg at a position corresponding toresidue 192, and a Thr, Ala, Met, or Val at a position corresponding toresidue 193. In some embodiments, an HA polypeptide variant (e.g., an H5HA polypeptide variant) has an Asp at a position corresponding toresidue 188, an Arg at a position corresponding to residue 192, and aThr, Ala, Met, or Val at a position corresponding to residue 193. Insome embodiments, an HA polypeptide variant (e.g., an H5 HA polypeptidevariant) has a Glu at a position corresponding to residue 188, a Lys ata position corresponding to residue 192, and a Thr, Ala, Met, or Val ata position corresponding to residue 193. In some embodiments, an HApolypeptide variant (e.g., an H5 HA polypeptide variant) has an Asp at aposition corresponding to residue 188, a Lys at a position correspondingto residue 192, and a Thr, Ala, Met, or Val at a position correspondingto residue 193.

In some embodiments, an HA polypeptide (e.g., an H5 HA polypeptide) hasan Ala at a position corresponding to residue 131. In some embodiments,an HA polypeptide variant (e.g., an H5 HA polypeptide variant) has a Thrat a position corresponding to residue 131.

In some embodiments, an HA polypeptide (e.g., an H5 HA polypeptide) hasa Ser at a position corresponding to residue 132. In some embodiments,an HA polypeptide variant (e.g., an H5 HA polypeptide variant) has a Thrat a position corresponding to residue 132.

In some embodiments, an HA polypeptide (e.g., an H5 HA polypeptide) hasa Ser at a position corresponding to residue 133. In some embodiments,an HA polypeptide variant (e.g., an H5 HA polypeptide variant) has a Thrat a position corresponding to residue 133.

In some embodiments, an HA polypeptide (e.g., an H5 HA polypeptide)includes Ala, Thr, and/or Ser at any position corresponding to residues131, 132, and/or 133. In some embodiments, an HA polypeptide variant(e.g., an H5 HA polypeptide variant) includes Ala, Thr, and/or Ser atany position corresponding to residues 131, 132, and/or 133. In someembodiments, an HA polypeptide variant (e.g., an H5 HA polypeptidevariant) includes Thr at all of positions corresponding to 131, 132, and133.

In some embodiments, an HA polypeptide (e.g., an H5 HA polypeptide) hasa Val at a position corresponding to residue 135. In some embodiments,an HA polypeptide variant (e.g., an H5 HA polypeptide variant) has anyamino acid other than Val at a position corresponding to residue 135.

In some embodiments, an HA polypeptide (e.g., an H5 HA polypeptide) hasa Ser at a position corresponding to residue 137. In some embodiments,an HA polypeptide variant (e.g., an H5 HA polypeptide variant) has anArg at a position corresponding to residue 137.

In some embodiments, an HA polypeptide (e.g., an H5 HA polypeptide) hasan Ile at a position corresponding to residue 155. In some embodiments,an HA polypeptide variant (e.g., an H5 HA polypeptide variant) has a Thrat a position corresponding to residue 155. In some embodiments, an HApolypeptide (e.g., an H5 HA polypeptide) includes a Thr at a positioncorresponding to residue 155. In some embodiments, an HA polypeptidevariant (e.g., an H5 HA polypeptide variant) includes a Thr at aposition corresponding to residue 155.

In some embodiments, an HA polypeptide (e.g., an H5 HA polypeptide) hasa Ser at a position corresponding to residue 221. In some embodiments,an HA polypeptide variant (e.g., an H5 HA polypeptide variant) has a Proat a position corresponding to residue 221.

In some embodiments, an HA polypeptide (e.g., an H5 HA polypeptide)includes a Ser at a position corresponding to residue 221. In someembodiments, an HA polypeptide variant (e.g., an H5 HA polypeptidevariant) includes a Pro at a position corresponding to residue 221.Without wishing to be bound by any one particular theory, Pro221 mightinfluence conformation of 220 loop which is involved with the RBS of H2HA.

In some embodiments, an HA polypeptide (e.g., an H5 HA polypeptide) hasa Gln at a position corresponding to residue 226. In some embodiments,an HA polypeptide variant (e.g., an H5 HA polypeptide variant) has a Leuat a position corresponding to residue 226.

In some embodiments, an HA polypeptide (e.g., an H5 HA polypeptide) hasa Ser at a position corresponding to residue 227. In some embodiments,an HA polypeptide variant (e.g., an H5 HA polypeptide variant) has a Glyat a position corresponding to residue 227.

In some embodiments, an HA polypeptide (e.g., an H5 HA polypeptide) hasa Gly at a position corresponding to residue 228. In some embodiments,an HA polypeptide variant (e.g., an H5 HA polypeptide variant) has a Serat a position corresponding to residue 228.

In some embodiments, an HA polypeptide (e.g., an H5 HA polypeptide)includes Gln, Ser, and Gly residues at positions corresponding toresidues 226, 227, and 228, respectively. In some embodiments, an HApolypeptide variant (e.g., an H5 HA polypeptide variant) includes a Leu,Gly, and Ser at positions corresponding to residues 226, 227, and 228,respectively.

In some embodiments, an HA polypeptide variant in accordance with theinvention, and particularly an H5 variant has one or more of thefollowing amino acids at the indicated positions (the numbering of thesepositions corresponds to the numbering of H3 HA):

-   -   Glu190Asp, Lys193Ser, Gly225Asp, Gln226Leu    -   Glu190Asp, Lys193Ser, Gln226Leu, Gly228Ser    -   Ala189Gln, Lys193Ser, Thr160Ala    -   Ala189Gln, Lys193Ser, Gln226Leu, Gly228Ser    -   Asp187Ser/Thr, Ala189Gln, Lys193Ser, Gln226Leu, Gly228Ser    -   Ala189Lys, Lys193Asn, Gln226Leu, Gly228Ser    -   Asp187Ser/Thr, Ala189Lys, Lys193Asn, Gln226Leu, Gly228Ser    -   Lys156Glu, Ala189Lys, Lys193Asn, Gln226Leu, Gly228Ser    -   Lys193His, Gln226Leu/Ile/Val, Gly228Ser    -   Lys193Arg, Gln226Leu/Ile/Val, Gly228Ser    -   Ala189Lys, Lys193Asn, Gly225Asp    -   Lys156Glu, Ala189Lys, Lys193Asn, Gly225Asp    -   Ser137Ala, Lys156Glu, Ala189Lys, Lys193Asn, Gly225Asp    -   Glu190Thr, Lys193Ser, Gly225Asp    -   Asp187Thr, Ala189Thr, Glu190Asp, Lys193Ser, Gly225Asp    -   Asn186Pro, Asp187Thr, Ala189Thr, Glu190Asp, Lys193Ser, Gly225Asp    -   Asn186Pro, Asp187Thr, Ala189Thr, Glu190Asp, Lys193Ser,        Gly225Asp, Ser227Ala    -   Gln226Leu, Gly228Ser, Thr160Ala    -   Gln226Leu, Gly228Ser, Thr160Ala    -   Gly228Ser, Thr160Ala    -   Gln226Leu, Thr160Ala    -   Gln226Leu, Gly228Ser    -   Thr160Ala    -   Gln226Leu, Gly228Ser, Asn158Xaa (wherein Xaa=any amino acid        besides Asn)    -   Gly228Ser, Asn158Xaa    -   Gln226Leu, Asn158Xaa    -   Gln226Leu, Gly228Ser    -   Asn158Xaa    -   Δ130 (wherein “Δ130” indicates a deletion at an amino acid        corresponding to position 130) plus any possible combination of        mutations at positions corresponding to residues 131, 132, 133,        135, 137, 155, 188, 192, 193, 221, 226, 227, and 228    -   Δ130 plus any possible combination of mutations at positions        corresponding to residues 131, 132, 135, 188, 192, and 221    -   Δ130 plus any possible combination of mutations at positions        corresponding to residues 133, 137, 155, 193, 226, 227, and 228    -   Δ130 plus any possible combination of mutations at positions        corresponding to residues 131, 132, 133, 135, 137, 155, 158,        160, 188, 192, 193, 221, 226, 227, and 228    -   Δ130 plus any possible combination of mutations at positions        corresponding to residues 131, 133, 137, 155, 188, 192, 193,        226, 227, and 228    -   Δ130 plus any possible combination of mutations at positions        corresponding to residues 131, 133, 137, 155, 188, 192, 193,        226, and 228    -   Δ130 plus any possible combination of mutations at positions        corresponding to residues 131, 133, 137, 155, 159, 160, 188,        192, 193, 226, 227, and 228    -   Δ130 plus any possible combination of mutations at positions        corresponding to residues 131, 133, 137, 155, 159, 160, 188,        192, 193, 226, and 228    -   Δ130 plus any possible combination of mutations at positions        corresponding to residues 137, 188, 192, 193, 226, 228, 131,        132, 133, 221, and 227    -   Δ130 plus any possible combination of mutations at positions        corresponding to residues 131, 132, 133, 221, and 227    -   Δ130 plus any possible combination of mutations at positions        corresponding to residues 137, 188, 192, 193, 226, and 228    -   Δ130 plus any possible combination of mutations at positions        corresponding to residues 137, 188, 192, 193, 226, 227, 228,        131, 132, and 133    -   Δ130 plus any possible combination of mutations at positions        corresponding to residues 227, 131, 132, and 133    -   Gln226Leu, Gly228Ser, Thr160Ala, Δ130    -   Gln226Leu, Gly228Ser, Δ130    -   Gln226Leu, Thr160Ala, Δ130    -   Gly228Ser, Thr160Ala, Δ130    -   Gln226Leu, Δ130    -   Gly228Ser, Δ130    -   Thr160Ala, Δ130    -   Δ130    -   Δ130, Ala131Thr, Leu133Thr, Ser137Arg, Ile155Thr, Ala188Glu,        Thr/Ile192Arg/Lys, Arg/Lys193Thr/Ala, Gln226Leu, Ser227Gly,        Gly228Ser    -   Δ130, Ala131Thr, Leu133Thr, Ser137Arg, Ile155Thr, Ala188Glu,        Thr/Ile192Arg/Lys, Arg/Lys193Thr/Ala, Gln226Leu, Gly228Ser    -   Δ130, Ala131Thr, Leu133Thr, Ser137Arg, Ile155Thr, Asn159Asp (or        Thr160Ala or both), Ala188Glu, Thr/Ile192Arg/Lys,        Arg/Lys193Thr/Ala, Gln226Leu, Ser227Gly, Gly228Ser    -   Δ130, Ala131Thr, Leu133Thr, Ser137Arg, Ile155Thr, Asn159Asp (or        Thr160Ala or both), Ala188Glu, Thr/Ile192Arg/Lys,        Arg/Lys193Thr/Ala, Gln226Leu, Gly228Ser    -   Δ130, Ser137Arg, Ala188Glu, Thr192Arg/Lys,        Arg/Lys193Thr/Met/Ala/Val, Gln226Leu, Gly228Ser    -   Δ130, Ser137Arg, Ala188Glu, Thr192Arg/Lys,        Arg/Lys193Thr/Met/Ala/Val, Gln226Leu, Gly228Ser, Xaa131Ser/Thr,        Xaa132Ser/Thr, Xaa133Ser/Thr, Ser221Pro, Ser227Gly (wherein        Xaa=any amino acid)    -   Δ130, Xaa131Ser/Thr, Xaa132Ser/Thr, Xaa133Ser/Thr, Ser221Pro,        Ser227Gly (wherein Xaa=any amino acid)    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid)    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue)    -   Δ130, Xaa193Xaa′ (wherein Xaa=a basic residue, e.g., Lys or Arg,        and Xaa′=a neutral or acidic residue)    -   Δ130, Lys/Arg193Thr/Ala/Met/Val    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid), Xaa193Xaa′ (wherein Xaa=a        basic residue, e.g., Lys or Arg, and Xaa′=a neutral or acidic        residue)    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue),        Xaa193Xaa′ (wherein Xaa=a basic residue, e.g., Lys or Arg, and        Xaa′=a neutral or acidic residue)    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid), Lys/Arg193Thr/Ala/Met/Val    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue),        Lys/Arg193Thr/Ala/Met/Val    -   Δ130, Ala188Glu    -   Δ130, Ala188Asp    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid), Ala188Glu    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue),        Ala188Glu    -   Δ130, Xaa193Xaa′ (wherein Xaa=a basic residue, e.g., Lys or Arg,        and Xaa′=a neutral or acidic residue), Ala188Glu    -   Δ130, Lys/Arg193Thr/Ala/Met/Val, Ala188Glu    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid), Ala188Asp    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue),        Ala188Asp    -   Δ130, Xaa193Xaa′ (wherein Xaa=a basic residue, e.g., Lys or Arg,        and Xaa′=a neutral or acidic residue), Ala188Asp    -   Δ130, Lys/Arg193Thr/Ala/Met/Val, Ala188Asp    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid), Xaa193Xaa′ (wherein Xaa=a        basic residue, e.g., Lys or Arg, and Xaa′=a neutral or acidic        residue), Ala188Glu    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue),        Xaa193Xaa′ (wherein Xaa=a basic residue, e.g., Lys or Arg, and        Xaa′=a neutral or acidic residue), Ala188Glu    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid), Lys/Arg193Thr/Ala/Met/Val,        Ala188Glu    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue),        Lys/Arg193Thr/Ala/Met/Val, Ala188Glu    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid), Xaa193Xaa′ (wherein Xaa=a        basic residue, e.g., Lys or Arg, and Xaa′=a neutral or acidic        residue), Ala188Asp    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue),        Xaa193Xaa′ (wherein Xaa=a basic residue, e.g., Lys or Arg, and        Xaa′=a neutral or acidic residue), Ala188Asp    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid), Lys/Arg193Thr/Ala/Met/Val,        Ala188Asp    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue),        Lys/Arg193Thr/Ala/Met/Val, Ala188Asp

In some embodiments, the present invention provides HA polypeptides(e.g., HA polypeptide variants, engineered HA polypeptides, and/orengineered HA polypeptide variants) whose amino acid sequence includesan element as set forth below:

-   -   X190, X193, X225 and X226    -   X190, X193, X226 and X228    -   X189, X193, X160    -   X189, X193, X226, X228    -   X187, X189, X193, X226, X228    -   X189, X193, X226, X228    -   X187, X189, X193, X226, X228    -   X156, X189, X193, X226, X228    -   X193, X226, X228    -   X193, X226, X228    -   X189, X193, X225    -   X156, X189, X193, X225    -   X137, X156, X189, X193, X225    -   X190, X193, X225    -   X187, X189, X190, X193, X225    -   X186, X187, X189, X190, X193, X225    -   X186, X187, X189, X190, X193, X225, X227    -   X226, X228, X160    -   X226, X228, X160    -   X228, X160    -   X226, X160    -   X226, X228    -   X160    -   X226, X228, Xaa158 (wherein Xaa=any amino acid besides Asn)    -   X228, Xaa158 (wherein Xaa=any amino acid besides Asn)    -   X226, Xaa158 (wherein Xaa=any amino acid besides Asn)    -   X226, X228    -   X158 (wherein Xaa=any amino acid besides Asn)    -   X130 plus any possible combination of X131, X132, X133, X135,        X137, X155, X188, X192, X193, X221, X226, X227, and X228    -   X130 plus any possible combination of X131, X132, X135, X188,        X192, and X221    -   X130 plus any possible combination of X133, X137, X155, X193,        X226, X227, and X228    -   X130 plus any possible combination of X131, X132, X133, X135,        X137, X155, Xaa158 (wherein Xaa=any amino acid besides Asn),        X160, X188, X192, X193, X221, X226, X227, and X228    -   X130 plus any possible combination of X131, X133, X137, X155,        X188, X192, X193, X226, X227, and X228    -   X130 plus any possible combination of X131, X133, X137, X155,        X188, X192, X193, X226, and X228    -   X130 plus any possible combination of X131, X133, X137, X155,        X159, X160, X188, X192, X193, X226, X227, and X228    -   X130 plus any possible combination of X131, X133, X137, X155,        X159, X160, X188, X192, X193, X226, and X228    -   X130 plus any possible combination of X137, X188, X192, X193,        X226, X228, X131, X132, X133, X221, and X227    -   X130 plus any possible combination of X131, X132, X133, X221,        and X227    -   X130 plus any possible combination of X137, X188, X192, X193,        X226, and X228    -   X130 plus any possible combination of X137, X188, X192, X193,        X226, X227, X228, X131, X132, and X133    -   X130 plus any possible combination of X227, X131, X132, and X133    -   X226, X228, X160, X130    -   X226, X228, X130    -   X226, X160, X130    -   X228, X160, X130    -   X226, X130    -   X228, X130    -   X160, X130    -   X130    -   X130, X131, X133, X137, X155, X188, X192, X193, X226, X227, X228    -   X130, X131, X133, X137, X155, X188, X192, X193, X226, X228    -   X130, X131, X133, X137, X155, X159, X160, X188, X192, X193,        X226, X227, X228    -   X130, X131, X133, X137, X155, X159, X160, X188, X192, X193,        X226, X228    -   X130, X137, X188, X192, X193, X226, X228    -   X130, X137, X188, X192, X193, X226, X228, X131, X132, X133,        X221, X227    -   X130, X131, X132, X133, X221, X227    -   X130, X192    -   X130, X193    -   X130, X192, X193    -   X130, X188    -   X130, X192, X188    -   X130, X193, X188    -   X130, X192, X193, X188

wherein X=any amino acid (unless otherwise specified above), and/or X=amissing amino acid. The numbering of these positions corresponds to thenumbering of H3 HA.

In some embodiments X130 is a deletion at position 130. In someembodiments, X160 is an Ala. In some embodiments, X158 is any amino acidother than Asn.

In some such embodiments, the H5 HA polypeptide variant has at least onefurther substitution as compared with a wild type H5 HA, such thataffinity and/or specificity of the variant for umbrella glycans isincreased.

In some such embodiments, the HA polypeptide has at least one furthersubstitution as compared with a wild type HA, such that affinity and/orspecificity of the variant for umbrella glycans is increased.

In some embodiments, HA polypeptides in accordance with the invention(including H5 HA polypeptide variants) have sequences that include L226,S228, and A160. In some embodiments, HA polypeptides in accordance withthe invention (including H5 HA polypeptide variants) have sequences thatinclude L226 and A160. In some embodiments, HA polypeptides inaccordance with the invention (including H5 HA polypeptide variants)have sequences that include S228 and A160. In some embodiments, HApolypeptides in accordance with the invention (including H5 HApolypeptide variants) have sequences that include A160.

In some embodiments, HA polypeptides in accordance with the invention(including H5 HA polypeptide variants) have sequences that include L226,S228, and X158 (wherein X=any amino acid besides Asn). In someembodiments, HA polypeptides in accordance with the invention (includingH5 HA polypeptide variants) have sequences that include L226 and X158.In some embodiments, HA polypeptides in accordance with the invention(including H5 HA polypeptide variants) have sequences that include S228and X158. In some embodiments, HA polypeptides in accordance with theinvention (including H5 HA polypeptide variants) have sequences thatinclude X158.

In some embodiments, HA polypeptides in accordance with the invention(including H5 HA polypeptide variants) have sequences that include Δ130and any possible combination of mutations at positions corresponding toresidues 131, 132, 133, 135, 137, 155, 158, 160, 188, 192, 193, 221,226, 227, and 228.

In some embodiments, HA polypeptides in accordance with the invention(including H5 HA polypeptide variants) have sequences that include Δ130,L226, S228, A160 and any possible combination of mutations at positionscorresponding to residues 131, 132, 133, 135, 137, 155, 158, 188, 192,193, 221, and 227. In some embodiments, HA polypeptides in accordancewith the invention (including H5 HA polypeptide variants) have sequencesthat include Δ130, L226, A160, and any possible combination of mutationsat positions corresponding to residues 131, 132, 133, 135, 137, 155,158, 188, 192, 193, 221, 227, and 228. In some embodiments, HApolypeptides in accordance with the invention (including H5 HApolypeptide variants) have sequences that include Δ130, S228, A160, andany possible combination of mutations at positions corresponding toresidues 131, 132, 133, 135, 137, 155, 158, 188, 192, 193, 221, and 227.In some embodiments, HA polypeptides in accordance with the invention(including H5 HA polypeptide variants) have sequences that include Δ130,L226, S228, and any possible combination of mutations at positionscorresponding to residues 131, 132, 133, 135, 137, 155, 158, 160, 188,192, 193, 221, and 227.

In some embodiments, HA polypeptides in accordance with the invention(including H5 HA polypeptide variants) have sequences that include Δ130,A160, and any possible combination of mutations at positionscorresponding to residues 131, 132, 133, 135, 137, 155, 158, 188, 192,193, 221, 226, 227, and 228. In some embodiments, HA polypeptides inaccordance with the invention (including H5 HA polypeptide variants)have sequences that include Δ130, L226, and any possible combination ofmutations at positions corresponding to residues 131, 132, 133, 135,137, 155, 158, 160, 188, 192, 193, 221, and 227, and 228. In someembodiments, HA polypeptides in accordance with the invention (includingH5 HA polypeptide variants) have sequences that include Δ130, S228, andany possible combination of mutations at positions corresponding toresidues 131, 132, 133, 135, 137, 155, 158, 160, 188, 192, 193, 221,226, and 227.

In some embodiments, HA polypeptides in accordance with the invention(including H5 HA polypeptide variants) have sequences that include Δ130,L226, S228, X158 (wherein X =any amino acid besides Asn) and anypossible combination of mutations at positions corresponding to residues131, 132, 133, 135, 137, 155, 160, 188, 192, 193, 221, and 227. In someembodiments, HA polypeptides in accordance with the invention (includingH5 HA polypeptide variants) have sequences that include Δ130, L226,X158, and any possible combination of mutations at positionscorresponding to residues 131, 132, 133, 135, 137, 155, 160, 188, 192,193, 221, 227, and 228. In some embodiments, HA polypeptides inaccordance with the invention (including H5 HA polypeptide variants)have sequences that include Δ130, S228, X158, and any possiblecombination of mutations at positions corresponding to residues 131,132, 133, 135, 137, 155, 160, 188, 192, 193, 221, and 227. In someembodiments, HA polypeptides in accordance with the invention (includingH5 HA polypeptide variants) have sequences that include Δ130, L226,S228, and any possible combination of mutations at positionscorresponding to residues 131, 132, 133, 135, 137, 155, 158, 160, 188,192, 193, 221, and 227.

In some embodiments, HA polypeptides in accordance with the invention(including H5 HA polypeptide variants) have sequences that include Δ130,X158 (wherein X=any amino acid besides Asn), and any possiblecombination of mutations at positions corresponding to residues 131,132, 133, 135, 137, 155, 160, 188, 192, 193, 221, 226, 227, and 228.

In some embodiments, H5 HA polypeptide variants (e.g., H5 HA polypeptidevariants) in accordance with the invention have an open binding site ascompared with a parent HA, and particularly with a parent wild type HAs.

In some embodiments, HA polypeptides (e.g., H5 HA polypeptides) inaccordance with the invention bind to the following α2-6 sialylatedglycans:

and combinations thereof. In some embodiments, H5 HA polypeptides inaccordance with the invention bind to glycans of the structure:

and combinations thereof; and/or

and combinations thereof. In some embodiments, HA polypeptides (e.g., H5HA polypeptides) in accordance with the invention bind to

in some embodiments to

in some embodiments to

and in some embodiments to

In some embodiments, HA polypeptides (e.g., H5 HA polypeptides) inaccordance with the invention bind to umbrella topology glycans. In someembodiments, H5 HA polypeptides in accordance with the invention bind toat least some of the glycans (e.g., α2-6 sialylated glycans) depicted inFIGS. 9A-9B. In some embodiments, HA polypeptides (e.g., H5 HApolypeptides) in accordance with the invention bind to multiple glycansdepicted in FIGS. 9A-9B.

In some embodiments, HA polypeptides (e.g., H5 HA polypeptides) inaccordance with the invention bind to at least about 10%, about 15%,about 20%, about 25%, about 30% about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90% about 95%, or more of the glycans found on HAreceptors in human upper respiratory tract tissues (e.g., epithelialcells).

In one aspect, the present invention provides the particular recognitionthat high affinity binding to umbrella-topology glycans alone may not besufficient to confer effective transmission to/infectivity of humans.The present invention provides the insight that reduced binding tocone-topology glycans may also be important. In some embodiments, highaffinity binding to umbrella-topology glycans and reduced affinitybinding to cone-topology glycans may be involved in conferring effectivetransmission to/infectivity of humans. In some embodiments, highaffinity binding to umbrella-topology glycans is sufficient to confereffective transmission to/infectivity of humans. In some embodiments,high affinity binding to umbrella-topology glycans is sufficient toconfer effective transmission to/infectivity of humans, even if theaffinity of binding to cone-topology glycans is not reduced (e.g.,unchanged, increased, etc.).

In some embodiments, increased affinity and/or specificity of binding ofan HA polypeptide variant (e.g., an H5 HA polypeptide variant) toumbrella-topology glycans and reduced affinity and/or specificitybinding to cone-topology glycans may be involved in increasing orenhancing transmission to/infectivity of humans relative to a referencepolypeptide (e.g., the HA polypeptide variant's cognate parent HApolypeptide). In some embodiments, increased affinity and/or specificityof binding of an HA polypeptide variant (e.g., an H5 HA polypeptidevariant) to umbrella-topology glycans is sufficient to increase orenhance transmission to/infectivity of humans relative to a referencepolypeptide (e.g., the HA polypeptide variant's cognate parent HApolypeptide). In some embodiments, increased affinity and/or specificityof binding of an HA polypeptide variant (e.g., an H5 HA polypeptidevariant) to umbrella-topology glycans is sufficient to increase orenhance transmission to/infectivity of humans relative to a referencepolypeptide (e.g., the HA polypeptide variant's cognate parent HApolypeptide), even if the affinity and/or specificity of binding tocone-topology glycans is not reduced (e.g., unchanged, increased, etc.).In some embodiments, increased affinity and/or specificity of binding ofan HA polypeptide variant (e.g., an H5 HA polypeptide variant) toumbrella-topology glycans is sufficient to increase or enhancetransmission to/infectivity of humans relative to a referencepolypeptide (e.g., the HA polypeptide variant's cognate parent HApolypeptide), even if the affinity and/or specificity of binding tocone-topology glycans is equal to and/or greater than that of theaffinity and/or specificity of binding to umbrella-topology glycans.

Portions or Fragments of HA Polypeptides

The present invention further provides characteristic portions (whichmay or may not be binding agents) of HA polypeptides in accordance withthe invention and nucleic acids that encode them. In general, acharacteristic portion is one that contains a continuous stretch ofamino acids, or a collection of continuous stretches of amino acids,that together are characteristic of the HA polypeptide. Each suchcontinuous stretch generally will contain at least two amino acids.Furthermore, those of ordinary skill in the art will appreciate thattypically at least 5, at least 10, at least 15, at least 20 or moreamino acids are required to be characteristic of a H5 HA polypeptide. Ingeneral, a characteristic portion is one that, in addition to thesequence identity specified above, shares at least one functionalcharacteristic with the relevant intact HA polypeptide. In someembodiments, characteristic portions of HA polypeptides in accordancewith the invention share glycan binding characteristics with therelevant full-length HA polypeptides.

Non-HA Polypeptides

In some embodiments, binding agents provided in accordance with thepresent invention are polypeptides whose amino acid sequence does notinclude a characteristic HA sequence. Such polypeptides are referred toherein as “Non-HA polypeptides”. In some embodiments, a Non-HApolypeptide has an amino acid sequence selected in advance (e.g., viarational design, including for example, introduction of strategic aminoacid alterations [e.g., additions, deletions, and/or substitutions] ascompared with a reference sequence). In some embodiments, a Non-HApolypeptide has an amino acid sequence that is determined stochasticallyand, for example, identified on the basis of the desirable bindingcharacteristics defined herein.

Antibodies

In some embodiments, binding agents provided in accordance with thepresent invention are antibodies (e.g., that bind to umbrella topologyglycans and/or to umbrella topology glycan mimics). Antibodies suitablefor the invention include antibodies or fragments of antibodies thatbind immunospecifically to any umbrella topology glycan epitope. As usedherein, the term “antibodies” is intended to include immunoglobulins andfragments thereof which are specifically reactive to the designatedprotein or peptide, or fragments thereof. Suitable antibodies include,but are not limited to, human antibodies, primatized antibodies,chimeric antibodies, bi-specific antibodies, humanized antibodies,conjugated antibodies (i.e., antibodies conjugated or fused to otherproteins, radiolabels, cytotoxins), Small Modular ImmunoPharmaceuticals(“SMIPs™”), single chain antibodies, cameloid antibodies, and antibodyfragments. As used herein, the term “antibodies” also includes intactmonoclonal antibodies, polyclonal antibodies, single domain antibodies(e.g., shark single domain antibodies (e.g., IgNAR or fragmentsthereof)), multispecific antibodies (e.g. bi-specific antibodies) formedfrom at least two intact antibodies, and antibody fragments so long asthey exhibit the desired biological activity. Antibody polypeptides foruse herein may be of any type (e.g., IgA, IgD, IgE, IgG, IgM).

As used herein, an “antibody fragment” includes a portion of an intactantibody, such as, for example, the antigen-binding or variable regionof an antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)2, and Fv fragments; triabodies; tetrabodies; linear antibodies;single-chain antibody molecules; and multi specific antibodies formedfrom antibody fragments. The term “antibody fragment” also includes anysynthetic or genetically engineered protein that acts like an antibodyby binding to a specific antigen to form a complex. For example,antibody fragments include isolated fragments, “Fv” fragments,consisting of the variable regions of the heavy and light chains,recombinant single chain polypeptide molecules in which light and heavychain variable regions are connected by a peptide linker (“ScFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

Antibodies can be generated using methods well known in the art. Forexample, protocols for antibody production are described by Harlow andLane, 1988, Antibodies: A Laboratory Manual; incorporated herein byreference. Typically, antibodies can be generated in mouse, rat, guineapig, hamster, camel, llama, shark, or other appropriate host.Alternatively, antibodies may be made in chickens, producing IgYmolecules (Schade et al., 1996, ALTEX 13(5):80-85; incorporated hereinby reference). In some embodiments, antibodies suitable for the presentinvention are subhuman primate antibodies. For example, generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., international patentpublication number WO 91/11465, 1991; and in Losman et al., 1990, Int.J. Cancer 46:310; both of which are incorporated herein by reference).In some embodiments, monoclonal antibodies may be prepared usinghybridoma methods (Milstein and Cuello, 1983, Nature 305(5934):537-40;incorporated herein by reference). In some embodiments, monoclonalantibodies may also be made by recombinant methods (U.S. Pat. No.4,166,452, 1979; incorporated herein by reference).

In some embodiments, antibodies suitable for the invention may includehumanized or human antibodies. Humanized forms of non-human antibodiesare chimeric Igs, Ig chains or fragments (such as Fv, Fab, Fab′, F(ab′)2or other antigen-binding subsequences of Abs) that contain minimalsequence derived from non-human Ig. Generally, a humanized antibody hasone or more amino acid residues introduced from a non-human source.These non-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization is accomplished by substituting rodent complementaritydetermining regions (CDRs) or CDR sequences for the correspondingsequences of a human antibody (Riechmann et al., 1988, Nature332(6162):323-7; Verhoeyen et al., 1988, Science. 239(4847):1534-6; bothof which are incorporated herein by reference). Such “humanized”antibodies are chimeric Abs (U.S. Pat. No. 4,816,567, 1989; incorporatedherein by reference), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In some embodiments, humanized antibodies aretypically human antibodies in which some CDR residues and possibly someFR residues are substituted by residues from analogous sites in rodentAbs. Humanized antibodies include human Igs (recipient antibody) inwhich residues from a CDR of the recipient are replaced by residues froma CDR of a non-human species (donor antibody) such as mouse, rat orrabbit, having the desired specificity, affinity and capacity. In someinstances, corresponding non-human residues replace Fv frameworkresidues of the human Ig. Humanized antibodies may comprise residuesthat are found neither in the recipient antibody nor in the imported CDRor framework sequences. In general, the humanized antibody comprisessubstantially all of at least one, and typically two, variable domains,in which most if not all of the CDR regions correspond to those of anon-human Ig and most if not all of the FR regions are those of a humanIg consensus sequence. The humanized antibody optimally also comprisesat least a portion of an Ig constant region (Fc), typically that of ahuman Ig (Riechmann et al., 1988, Nature 332(6162):323-7; Verhoeyen etal., 1988, Science. 239(4847):1534-6; both of which are incorporatedherein by reference).

Human antibodies can also be produced using various techniques,including phage display libraries (Hoogenboom et al., 1991, Mol Immunol.28(9):1027-37; Marks et al., 1991, J Mol Biol. 222(3):581-97; both ofwhich are incorporated herein by reference) and the preparation of humanmonoclonal antibodies (Reisfeld and Sell, 1985, Cancer Surv.4(1):271-90; incorporated herein by reference). Similarly, introducinghuman Ig genes into transgenic animals in which the endogenous Ig geneshave been partially or completely inactivated can be exploited tosynthesize human antibodies. Upon challenge, human antibody productionis observed, which closely resembles that seen in humans in allrespects, including gene rearrangement, assembly, and antibodyrepertoire (Fishwild et al., 1996, Nat Biotechnol. 14(7):845-51; Lonberget al., 1994, Nature 368(6474):856-9; Lonberg and Huszar, 1995, Int.Rev. Immunol. 13(1):65-93; Marks et al., 1992, Biotechnology (N Y).10(7):779-83; all of which are incorporated herein by reference).

Lectins

In some embodiments, binding agents provided in accordance with thepresent invention are lectins. Lectins are sugar-binding proteins whichmay bind to a soluble carbohydrate or to a carbohydrate moiety which isa part of a glycoconjugate (e.g., a glycopeptide or glycolipid). Lectinstypically agglutinate certain animal cells and/or precipitateglycoconjugates by recognizing a particular sugar moiety. For example,SNA-1 is a lectin that has a high affinity for α2-6 sialic acids. As yetanother example, polyporus squamosus lectins (PSL1a and PSL1b) have highaffinity for binding sialylated glycoconjugates containingNeu5Acα2,6Galβ1,4Glc/GlcNAc trisaccharide sequences of asparagine-linkedglycoproteins. Non-limiting exemplary lectins that may act as bindingagents include SNA-1, SNA-1′, PSL1a, PSL1b, and polypeptides derivedtherefrom.

Amino acid sequences of exemplary lectins are provided below:

Sambucus Nigra Lectin 1 (Genbank Accession No. U27122): (SEQ ID NO: 56)MRLVAKLLYLAVLAICGLGIHGALTHPRVTPPVYPSVSFNLTGADTYEPFLRALQEKVILGNHTAFDLPVLNPESQVSDSNRFVLVPLTNPSGDTVTLAIDVVNLYVVAFSSNGKSYFFSGSTAVQRDNLFVDTTQEELNFTGNYTSLERQVGFGRVYIPLGPKSLDQAISSLRTYTLTAGDTKPLARGLLVVIQMVSEAARFRYIELRIRTSITDASEFTPDLLMLSMENNWSSMSSEIQQAQPGGIFAGVVQLRDERNNSIEVTNFRRLFELTYIAVLLYGCAPVTSSSYSNNAIDAQIIKMPVFRGGEYEKVCSVVEVTRRISGWDGLCVDVRYGHYIDGNPVQLRPCGNECNQLWTFRTDGTIRWLGKCLTASSSVMIYDCNTVPPEATKWVVSIDGTITNPHSGLVLTAPQAAEGTALSLENNIHAARQGWTVGDVEPLVTFIVGYKQMCLRENGENNFVWLEDCVLNRVQQEWALYGDGTIRVNSNRSLCVTSEDHEPSDLIVILKCEGSGNQRWVFNTNGTISNPNAKLLMDVAQRDVSLRKI ILYRPTGNPNQQWITTTHPASambucus Nigra Lectin 1′ (Genbank Accession No. U66191): (SEQ ID NO: 57)MKVVATILYLVVLAICGLGIHGAHPTHSAPPTVYPSVSFNLTEANSNEYRHFLQELRGKVILGSHRAFDLPVLNPESKVSDSDRFVLVRLTNPSRKKVTLAIDVVTFYVVAFAQNDRSYFFSGSSEVQRENLFVDTTQEDLNFKGDYTSLEHQVGFGRVYIPLGPKSLAQSISSLSTYKSSAGDNKRLARSLLVVIQMVSEAARFRYIQLRIQASITDAKEFTPDLLMLSMENKWSSMSSEIQQAQPGGAFAQVVKLLDQRNHPIDVTNFRRLFQLTSVAVLLHGCPTVTKMPAYIIKMPVFNGGEDEERCSVVEEVTRRIGGRDGFCAEVKNGDEKDGTPVQLSSCGEQSNQQWTFSTDGTIQSLGKCLTTSSSVMIYNCKVVPPESTKWVVSIDGTITNPRSGLVLTAPKAAEGTLVSLEKNVHAARQGWIVGNVEPLVTFIVGYEQMCLETNPGNNDVSLGDCSVKSASKVDQKWALYGDGTIRVNNDRSLCVTSEGKSSNEPIIILKCLGWANQRWVFNTDGTISNPDSKLVMHVDQNDVPLRKII LSHPSGTSNQQWIASTHPAPolyporous squamosus lectin 1a (UniProt Q75WT9) (SEQ ID NO: 58)MSFQGHGIYYIASAYVANTRLALSEDSSANKSPDVIISSDAVDPLNNLWLIEPVGEADTYTVRNAFAGSYMDLAGHAATDGTAIIGYRPTGGDNQKWIISQINDVWKIKSKETGTFVTLLNGDGGGTGTVVGWQNITNNTSQNWTFQKLSQTGANVHATLLACPALRQDFKSYLSDGLYLVLTRDQISSIWQASGLGSTPWRSEIFDCDDFATVFKGAVAKWGNENFKANGFALLCGLMFGSKSSGAHAYNWFVERGNFSTVTFFEPQNGTYSANAWDYKAYFGLFPolyporous squamosus lectin 1b (UniProt Q75WT8) (SEQ ID NO: 59)MSFEGHGIYHIPHAHVANIRMALANRGSGQNGTPVIAWDSNNDAFDHMWLVEPTGEADTYTIHNVSTGTYMDVTASAVADNTPIIGYQRTGNDNQKWIIRQVQTDGGDRPWKIQCKATGTFATLYSGGGSGTAIVGWRLVNSNGNQDWVFQKLSQTSVNVHATLLACGATVGQDFKNYLYDGLYLVLPRDRISAIWKASGLGETARRDGIYDSDEFAMTFKSAAATWGKENFKADGFAILCGMMFGTKASTNRHAYNWVVERGSFSTVTFFEPQNGTYSDDAWGYKAYFGLF

Aptamers

In some embodiments, binding agents provided in accordance with thepresent invention are aptamers. Aptamers are macromolecules composed ofnucleic acid (e.g., RNA, DNA) that bind tightly to a specific moleculartarget (e.g., an umbrella topology glycan). A particular aptamer may bedescribed by a linear nucleotide sequence and is typically about 15 toabout 60 nucleotides in length. Without wishing to be bound by anytheory, it is contemplated that the chain of nucleotides in an aptamerform intramolecular interactions that fold the molecule into a complexthree-dimensional shape, and this three-dimensional shape allows theaptamer to bind tightly to the surface of its target molecule. Given theextraordinary diversity of molecular shapes that exist within theuniverse of all possible nucleotide sequences, aptamers may be obtainedfor a wide array of molecular targets, including proteins and smallmolecules. In addition to high specificity, aptamers have very highaffinities for their targets (e.g., affinities in the picomolar to lownanomolar range for proteins). Aptamers are chemically stable and can beboiled or frozen without loss of activity. Because they are syntheticmolecules, they are amenable to a variety of modifications, which canoptimize their function for particular applications. For example,aptamers can be modified to dramatically reduce their sensitivity todegradation by enzymes in the blood for use in in vivo applications. Inaddition, aptamers can be modified to alter their biodistribution orplasma residence time.

Selection of aptamers that can bind umbrella topology glycans (and/or toumbrella topology glycan mimics) can be achieved through methods knownin the art. For example, aptamers can be selected using the SELEX(Systematic Evolution of Ligands by Exponential Enrichment) method(Tuerk and Gold, 1990, Science 249:505-510; incorporated herein byreference). In the SELEX method, a large library of nucleic acidmolecules (e.g., 10¹⁵ different molecules) is produced and/or screenedwith the target molecule (e.g., an umbrella topology glycan of umbrellatopology glycan epitope). The target molecule is allowed to incubatewith the library of nucleotide sequences for a period of time. Severalmethods, known in the art, can then be used to physically isolate theaptamer target molecules from the unbound molecules in the mixture,which can be discarded. The aptamers with the highest affinity for thetarget molecule can then be purified away from the target molecule andamplified enzymatically to produce a new library of molecules that issubstantially enriched for aptamers that can bind the target molecule.The enriched library can then be used to initiate a new cycle ofselection, partitioning, and amplification. After 5-15 cycles of thisiterative selection, partitioning and amplification process, the libraryis reduced to a small number of aptamers that bind tightly to the targetmolecule. Individual molecules in the mixture can then be isolated,their nucleotide sequences determined, and their properties with respectto binding affinity and specificity measured and compared. Isolatedaptamers can then be further refined to eliminate any nucleotides thatdo not contribute to target binding and/or aptamer structure, therebyproducing aptamers truncated to their core binding domain. See Jayasena,1999, Clin. Chem. 45:1628-50 for review of aptamer technology; theentire teachings of which are incorporated herein by reference).

Production of Polypeptides

Polypeptides in accordance with the invention (e.g., HA polypeptidesand/or Non-HA polypeptides), and/or characteristic portions thereof, ornucleic acids encoding them, may be produced by any available means.

Polypeptides in accordance with the invention (or characteristicportions thereof) may be produced, for example, by utilizing a host cellsystem engineered to express a polypeptide-encoding nucleic acid inaccordance with the invention.

Any system can be used to produce polypeptides (or characteristicportions), such as egg, baculovirus, plant, yeast, Madin-Darby CanineKidney cells (MDCK), or Vero (African green monkey kidney) cells.Alternatively or additionally, polypeptides (or characteristic portions)can be expressed in cells using recombinant techniques, such as throughthe use of an expression vector (Sambrook et al., Molecular Cloning: ALaboratory Manual, CSHL Press, 1989; incorporated herein by reference).

Alternatively or additionally, polypeptides in accordance with theinvention (or characteristic portions thereof) can be produced bysynthetic means.

Alternatively or additionally, polypeptides in accordance with theinvention (or characteristic portions thereof), and particularly HApolypeptides, may be produced in the context of intact virus, whetherotherwise wild type, attenuated, killed, etc. Polypeptides in accordancewith the invention (e.g., HA polypeptides), or characteristic portionsthereof, may also be produced in the context of virus like particles.

In some embodiments, HA polypeptides (or characteristic portionsthereof) can be isolated and/or purified from influenza virus. Forexample, virus may be grown in eggs, such as embryonated hen eggs, inwhich case the harvested material is typically allantoic fluid.Alternatively or additionally, influenza virus may be derived from anymethod using tissue culture to grow the virus. Suitable cell substratesfor growing the virus include, for example, dog kidney cells such asMDCK or cells from a clone of MDCK, MDCK-like cells, monkey kidney cellssuch as AGMK cells including Vero cells, cultured epithelial cells ascontinuous cell lines, 293T cells, BK-21 cells, CV-1 cells, or any othermammalian cell type suitable for the production of influenza virus forvaccine purposes, readily available from commercial sources (e.g., ATCC,Rockville, Md.). Suitable cell substrates also include human cells suchas MRC-5 cells. Suitable cell substrates are not limited to cell lines;for example primary cells such as chicken embryo fibroblasts are alsoincluded.

Also, it will be appreciated by those of ordinary skill in the art thatpolypeptides, and particularly variant HA polypeptides as describedherein, may be generated, identified, isolated, and/or produced byculturing cells or organisms that produce the polypeptide (whether aloneor as part of a complex, including as part of a virus particle orvirus), under conditions that allow ready screening and/or selection ofpolypeptides capable of binding to umbrella-topology glycans. To givebut one example, in some embodiments, it may be useful to produce and/orstudy a collection of polypeptides (e.g., HA variant polypeptides) underconditions that reveal and/or favor those variants that bind to umbrellatopology glycans (e.g., with particular specificity and/or affinity). Insome embodiments, such a collection of polypeptides (e.g., HA variantpolypeptides) results from evolution in nature. In some embodiments,such a collection of polypeptides (e.g., HA variant polypeptides)results from engineering. In some embodiments, such a collection ofpolypeptides (e.g., HA variant polypeptides) results from a combinationof engineering and natural evolution.

HA Receptors

HA interacts with the surface of cells by binding to a glycoproteinreceptor. Binding of HA to HA receptors is predominantly mediated byN-linked glycans on the HA receptors. Specifically, HA on the surface offlu virus particles recognizes sialylated glycans that are associatedwith HA receptors on the surface of the cellular host. After recognitionand binding, the host cell engulfs the viral cell and the virus is ableto replicate and produce many more virus particles to be distributed toneighboring cells. Some crystal structures of exemplary HA-glycaninteractions have been identified and are presented in Table 1:

TABLE 1 Crystal Structures of HA-Glycan Complexes Abbreviation (PDB ID)Virus Strain Glycan (with assigned coordinates) ADkALB76_H1_26 (2WRH)A/duck/Alberta/76 (H1N1) Neu5Ac ASI30_H1_23 (1RV0) A/Swine/Iowa/30(H1N1) Neu5Ac ASI30_H1_26 (1RVT) A/Swine/Iowa/30 (H1N1)Neu5Acα6Galβ4GlcNAcβ3Galβ4Glc ASC18_H1_26 (2WRG) A/South Carolina/1/18(H1N1) Neu5Acα6Galβ4GlcNAcβ3Gal APR34_H1_23 (1RVX) A/Puerto Rico/8/34(H1N1) Neu5Acα3Galβ4GlcNAc APR34_H1_26 (1RVZ) A/Puerto Rico/8/34 (H1N1)Neu5Acα6Galβ4GlcNAc ACkNY91_H2_23 (2WR2) A/chicken/NY/29878/91 (H2N2)Neu5Acα3Galβ3GlcNAc AckNY91_H2_26 (2WR1) A/chicken/NY/29878/91 (H2N2)Neu5Acα6Galβ4GlcNAc AdkON77_H2_23 (2WR3) A/duck/Ontario/77 (H2N2)Neu5Acα3Galβ4GlcNAc AdkON77_H2_26 (2WR4) A/duck/Ontario/77 (H2N2)Neu5Acα6Galβ4GlcNAc AckPD84_H2_26 (2WRF) A/chicken/Potsdam/475/84 (H2N2)Neu5Acα6Gal ASING57_H2_23 (2WRB) A/Singapore/1/57 (H2N2) Neu5AcASING57_H2_26 (2WR7) A/Singapore/1/57 (H2N2) Neu5Acα6Galβ4GlcNAcβ3GalAJAP57_H2_26(2WRE) A/Japan/305/57 (H2N2) Neu5Acα6Gal ADU63_H3_23 (1MQM)A/Duck/Ukraine/1/63 (H3N8) Neu5Acα3Gal ADU63_H3_26 (1MQN)A/Duck/Ukraine/1/63 (H3N8) Neu5Acα6Gal AAI68_H3_23 (1HGG) A/Aichi/2/68(H3N2) Neu5Acα3Galβ4Glc ADS97_H5_23 (1JSN) A/Duck/Singapore/3/97 (H5N3)Neu5Acα3Galβ3GlcNAc ADS97_H5_26(1JSO) A/Duck/Singapore/3/97 (H5N3)Neu5Ac Viet1203_04_H5 (2FK0) ANietnam/1203/2004 (H5N1) Viet1194_04_H5(2IBX) ANietnam/1194/2004 (H5N1) ASI30_H1_23 (1RV0) A/Swine/Iowa/30(H1N1) Neu5Ac ASI30_H1_26 (1RVT) A/Swine/Iowa/30 (H1N1)Neu5Acα6Galβ4GlcNAcβ3Galβ4Glc APR34_H123 (1RVX) A/Puerto Rico/8/34(H1N1) Neu5Acα3Galβ4GlcNAc APR34_H126 (1RVZ) A/Puerto Rico/8/34 (H1N1)Neu5Acα6Galβ4GlcNAc ADU63_H3_23 (1MQM) A/Duck/Ukraine/1/63 (H3N8)Neu5Acα3Gal ADU63_H3_26 (1MQN) A/Duck/Ukraine/1/63 (H3N8) Neu5Acα6GalAAI68_H3_23 (1HGG) A/Aichi/2/68 (H3N2) Neu5Acα3Galβ4Glc ADS97_H5_23(1JSN) A/Duck/Singapore/3/97 (H5N3) Neu5Acα3Galβ3GlcNAcADS97_H5_26(1JSO) A/Duck/Singapore/3/97 (H5N3) Neu5Ac Viet04_H5 (2FK0)ANietnam/1203/2004 (H5N1)HA-α2-6 sialylated glycan complexes were generated by superimposition ofthe CA trace of the HA1 subunit of ADU63_H3 and ADS97_H5 and Viet04_H5on ASI30_H1_26 and APR34_H1_26 (H1). Although the structural complexesof the human A/Aichi/2/68 (H3N2) with α2-6 sialylated glycans arepublished (Eisen et al, 1997, Virology, 232:19; incorporated herein byreference), their coordinates were not available in the Protein DataBank. The SARF2 program (available through the world wide web at123d.ncifcrf.gov/sarf2) was used to obtain the structural alignment ofthe different HA1 subunits for superimposition.

HA receptors are modified by either α2-3 or α2-6 sialylated glycans nearthe receptor's HA-binding site, and the type of linkage of thereceptor-bound glycan can affect the conformation of the receptor'sHA-binding site, thus affecting the receptor's specificity for differentHAs.

For example, the glycan binding pocket of avian HA is narrow. Accordingto the present invention, this pocket binds to the trans conformation ofα2-3 sialylated glycans, and/or to cone-topology glycans, whether α2-3or α2-6 linked.

HA receptors in avian tissues, and also in human deep lung andgastrointestinal (GI) tract tissues are characterized by α2-3 sialylatedglycan linkages, and furthermore (according to the present invention),are characterized by glycans, including α2-3 sialylated and/or α2-6sialylated glycans, which predominantly adopt cone topologies. HAreceptors having such cone-topology glycans may be referred to herein asCTHArs.

By contrast, human HA receptors in the bronchus and trachea of the upperrespiratory tract are modified by α2-6 sialylated glycans. Unlike theα2-3 motif, the α2-6 motif has an additional degree of conformationalfreedom due to the C6-C5 bond (Russell et al., 2006, Glycoconj J23:85;incorporated herein by reference). HAs that bind to such α2-6 sialylatedglycans have a more open binding pocket to accommodate the diversity ofstructures arising from this conformational freedom. Moreover, accordingto the present invention, HAs may need to bind to glycans (e.g., α2-6sialylated glycans) in an umbrella topology, and particularly may needto bind to such umbrella topology glycans with strong affinity and/orspecificity, in order to effectively mediate infection of human upperrespiratory tract tissues. HA receptors having umbrella-topology glycansmay be referred to herein as UTHArs.

As a result of these spatially restricted glycosylation profiles, humansare not usually infected by viruses containing many wild type avian HAs(e.g., avian H5). Specifically, because the portions of the humanrespiratory tract that are most likely to encounter virus (i.e., thetrachea and bronchi) lack receptors with cone glycans (e.g., α2-3sialylated glycans, and/or short glycans) and wild type avian HAstypically bind primarily or exclusively to receptors associated withcone glycans (e.g., α2-3 sialylated glycans, and/or short glycans),humans rarely become infected with avian viruses. Only when insufficiently close contact with virus that it can access the deep lungand/or gastrointestinal tract receptors having umbrella glycans (e.g.,long α2-6 sialylated glycans) do humans become infected.

Glycan Arrays

To rapidly expand the current knowledge of known specific glycan-glycanbinding protein (GBP) interactions, the Consortium for FunctionalGlycomics (CFG; available through the world wide web atfunctionalglycomics.org), an international collaborative researchinitiative, has developed glycan arrays comprising several glycanstructures that have enabled high throughput screening of GBPs for novelglycan ligand specificities. The glycan arrays comprise both monovalentand polyvalent glycan motifs (i.e. attached to polyacrylamide backbone),and each array comprises 264 glycans with low (10 μM) and high (100 μM)concentrations, and six spots for each concentration (see the world wideweb atfunctionalglycomics.org/static/consortium/resources/resourcecoreh5).

The arrays predominantly comprise synthetic glycans that capture thephysiological diversity of N- and O-linked glycans. In addition to thesynthetic glycans, N-linked glycan mixtures derived from differentmammalian glycoproteins are also represented on the array.

As used herein, a glycan “array” refers to a set of one or more glycans,optionally immobilized on a solid support. In some embodiments, an“array” is a collection of glycans present as an organized arrangementor pattern at two or more locations that are physically separated inspace. Typically, a glycan array will have at least 4, at least 8, atleast 16, at least 24, at least 48, at least 96, or several hundred orthousand discrete locations. In general, glycan arrays in accordancewith the invention may have any of a variety of formats. Variousdifferent array formats applicable to biomolecules are known in the art.For example, a huge number of protein and/or nucleic acid arrays arewell known. Those of ordinary skill in the art will immediatelyappreciate standard array formats appropriate for glycan arrays of thepresent invention.

In some embodiments, glycan arrays in accordance with the invention arepresent in “microarray” formats. A microarray may typically have samplelocations separated by a distance of about 50μ to about 200μ or less andimmobilized sample in the nano to micromolar range or nano to picogramrange. Array formats known in the art include, for example, those inwhich each discrete sample location has a scale of, for example, ten μ.

In some embodiments, glycan arrays in accordance with the inventioncomprise a plurality of glycans spatially immobilized on a support. Thepresent invention provides glycan molecules arrayed on a support. Asused herein, “support” refers to any material which is suitable to beused to array glycan molecules. As will be appreciated by those ofordinary skill in the art, any of a wide variety of materials may beemployed. To give but a few examples, support materials which may be ofuse in the invention include hydrophobic membranes, for example,nitrocellulose, PVDF or nylon membranes. Such membranes are well knownin the art and can be obtained from, for example, Bio-Rad, HemelHempstead, UK.

In some embodiments, the support on which glycans are arrayed maycomprise a metal oxide. Suitable metal oxides include, but are notlimited to, titanium oxide, tantalum oxide, and aluminum oxide. Examplesof such materials may be obtained from Sigma-Aldrich Company Ltd, FancyRoad, Poole, Dorset. BH12 4QH UK.

In some embodiments, such a support is or comprises a metal oxide gel. Ametal oxide gel is considered to provide a large surface area within agiven macroscopic area to aid immobilization of thecarbohydrate-containing molecules.

Additional or alternative support materials which may be used inaccordance with the present invention include gels, for example silicagels or aluminum oxide gels. Examples of such materials may be obtainedfrom, for example, Merck KGaA, Darmstadt, Germany.

In some embodiments, glycan arrays are immobilized on a support that canresist change in size or shape during normal use. For example a supportmay be a glass slide coated with a component material suitable to beused to array glycans. Also, some composite materials can desirableprovide solidity to a support.

As demonstrated herein, arrays in accordance with the invention areuseful for the identification and/or characterization of different HApolypeptides and their binding characteristics. In some embodiments, HApolypeptides in accordance with the invention are tested on such arraysto assess their ability to bind to umbrella topology glycans (e.g., toα2-6 sialylated glycans, and particularly to long α2-6 sialylatedglycans arranged in an umbrella topology).

Indeed, the present invention provides arrays of α2-6 sialylatedglycans, and optionally α2-3 sialylated glycans, that can be used tocharacterize HA polypeptide binding capabilities and/or as a diagnosticto detect, for example, human-binding HA polypeptides. In someembodiments, arrays in accordance with the invention contain glycans(e.g., α2-6 sialylated glycans, and particularly long α2-6 sialylatedglycans) in an umbrella topology. As will be clear to those of ordinaryskill in the art, such arrays are useful for characterizing or detectingany HA polypeptides, including for example, those found in naturalinfluenza isolates in addition to those designed and/or prepared byresearchers.

In some embodiments, such arrays include glycans representative of about10%, about 15%, about 20%, about 25%, about 30% about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90% about 95%, or more of the glycans(e.g., the umbrella glycans, which will often be α2-6 sialylatedglycans, particularly long α2-6 sialylated glycans) found on human HAreceptors, and particularly on human upper respiratory tract HAreceptors. In some embodiments, arrays in accordance with the inventioninclude some or all of the glycan structures depicted in FIG. 10 In someembodiments, arrays include at least about 10%, about 15%, about 20%,about 25%, about 30% about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95%, or more of these depicted glycans.

The present invention provides methods for identifying or characterizingHA proteins using glycan arrays. In some embodiments, for example, suchmethods comprise steps of (1) providing a sample containing HApolypeptide, (2) contacting the sample with a glycan array comprising,and (3) detecting binding of HA polypeptide to one or more glycans onthe array.

Suitable sources for samples containing HA polypeptides to be contactedwith glycan arrays according to the present invention include, but arenot limited to, pathological samples, such as blood, serum/plasma,peripheral blood mononuclear cells/peripheral blood lymphocytes(PBMC/PBL), sputum, urine, feces, throat swabs, dermal lesion swabs,cerebrospinal fluids, cervical smears, pus samples, food matrices, andtissues from various parts of the body such as brain, spleen, and liver.Alternatively or additionally, other suitable sources for samplescontaining HA polypeptides include, but are not limited to,environmental samples such as soil, water, and flora. Yet other samplesinclude laboratory samples, for example of engineered HA polypeptidesdesigned and/or prepared by researchers. Other samples that have notbeen listed may also be applicable.

A wide variety of detection systems suitable for assaying HA polypeptidebinding to glycan arrays in accordance with the invention are known inthe art. For example, HA polypeptides can be detectably labeled(directly or indirectly) prior to or after being contacted with thearray; binding can then be detected by detection of localized label. Insome embodiments, scanning devices can be utilized to examine particularlocations on an array.

Alternatively or additionally, binding to arrayed glycans can bemeasured using, for example, calorimetric, fluorescence, or radioactivedetection systems, or other labeling methods, or other methods that donot require labeling. In general, fluorescent detection typicallyinvolves directly probing the array with a fluorescent molecule andmonitoring fluorescent signals. Alternatively or additionally, arrayscan be probed with a molecule that is tagged (for example, with biotin)for indirect fluorescence detection (in this case, by testing forbinding of fluorescently-labeled streptavidin). Alternatively oradditionally, fluorescence quenching methods can be utilized in whichthe arrayed glycans are fluorescently labeled and probed with a testmolecule (which may or may not be labeled with a different fluorophore).In such embodiments, binding to the array acts to squelch thefluorescence emitted from the arrayed glycan, therefore binding isdetected by loss of fluorescent emission. Alternatively or additionally,arrayed glycans can be probed with a live tissue sample that has beengrown in the presence of a radioactive substance, yielding aradioactively labeled probe. Binding in such embodiments can be detectedby measuring radioactive emission.

Such methods are useful to determine the fact of binding and/or theextent of binding by HA polypeptides to glycan arrays in accordance withthe invention. In some embodiments, such methods can further be used toidentify and/or characterize agents that interfere with or otherwisealter glycan-HA polypeptide interactions.

Methods described below may be of particular use in, for example,identifying whether a molecule thought to be capable of interacting witha carbohydrate can actually do so, or to identify whether a moleculeunexpectedly has the capability of interacting with a carbohydrate.

The present invention also provides methods of using arrays inaccordance with the invention, for example, to detect a particular agentin a test sample. For instance, such methods may comprise steps of (1)contacting a glycan array with a test sample (e.g., with a samplethought to contain an HA polypeptide); and, (2) detecting the binding ofany agent in the test sample to the array.

Yet further, binding to arrays in accordance with the invention may beutilized, for example, to determine kinetics of interaction betweenbinding agent and glycan. For example, methods in accordance with theinvention for determining interaction kinetics may include steps of (1)contacting a glycan array with the molecule being tested; and, (2)measuring kinetics of interaction between the binding agent and arrayedglycan(s).

The kinetics of interaction of a binding agent with any of the glycansin an array in accordance with the invention can be measured by realtime changes in, for example, colorimetric or fluorescent signals, asdetailed above. Such methods may be of particular use in, for example,determining whether a particular binding agent is able to interact witha specific carbohydrate with a higher degree of binding than does adifferent binding agent interacting with the same carbohydrate.

It will be appreciated, of course, that glycan binding by HApolypeptides in accordance with the invention can be evaluated on glycansamples or sources not present in an array format per se. For example,HA polypeptides in accordance with the invention can be bound to tissuesamples and/or cell lines to assess their glycan bindingcharacteristics. Appropriate cell lines include, for example, any of avariety of mammalian cell lines, particularly those expressing HAreceptors containing umbrella topology glycans (e.g., at least some ofwhich may be α2-6 sialylated glycans, and particularly long α2-6sialylated glycans). In some embodiments, utilized cell lines expressindividual glycans with umbrella topology. In some embodiments, utilizedcell lines express a diversity of glycans. In some embodiments, celllines are obtained from clinical isolates; in some they are maintainedor manipulated to have a desired glycan distribution and/or prevalence.In some embodiments, tissue samples and/or cell lines express glycanscharacteristic of mammalian upper respiratory epithelial cells.

Nucleic Acids

In some embodiments, the present invention provides nucleic acids whichencode an HA polypeptide or a characteristic or biologically activeportion of an HA polypeptide. In some embodiments, the inventionprovides nucleic acids which are complementary to nucleic acids whichencode an HA polypeptide or a characteristic or biologically activeportion of an HA polypeptide.

In some embodiments, the invention provides nucleic acid molecules whichhybridize to nucleic acids encoding an HA polypeptide or acharacteristic or biologically active portion of an HA polypeptide. Suchnucleic acids can be used, for example, as primers or as probes. To givebut a few examples, such nucleic acids can be used as primers inpolymerase chain reaction (PCR), as probes for hybridization (includingin situ hybridization), and/or as primers for reverse transcription-PCR(RT-PCR).

In some embodiments, nucleic acids can be DNA or RNA, and can be singlestranded or double-stranded. In some embodiments, nucleic acids inaccordance with the invention may include one or more non-naturalnucleotides; in some embodiments, nucleic acids in accordance with theinvention include only natural nucleotides.

Antibodies to Polypeptides

The present invention provides antibodies to binding agent polypeptidesin accordance with the invention (e.g., HA polypeptides). These may bemonoclonal or polyclonal and may be prepared by any of a variety oftechniques known to those of ordinary skill in the art (e.g., see Harlowand Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory; incorporated herein by reference). For example, antibodiescan be produced by cell culture techniques, including the generation ofmonoclonal antibodies, or via transfection of antibody genes intosuitable bacterial or mammalian cell hosts, in order to allow for theproduction of recombinant antibodies.

Testing Binding Agents in Animal Models

The present invention provides methods for testing binding agents inaccordance with the invention (e.g., HA polypeptides, LSBAs, USBAs,UTSBAs, etc.) in an animal host. As used herein, an “animal host”includes any animal model suitable for influenza research. For example,animal hosts suitable for the invention can be any mammalian hosts,including primates, ferrets, cats, dogs, cows, horses, rodents such as,mice, hamsters, rabbits, and rats. In some embodiments, an animal hostused for the invention is a ferret. In particular, in some embodiments,an animal host is naïve to viral exposure or infection prior toadministration of a binding agent in accordance with the invention(optionally in a composition in accordance with the invention). In someembodiments, the animal host is inoculated with, infected with, orotherwise exposed to virus prior to or concurrent with administration ofa binding agent in accordance with the invention. An animal host used inthe practice of the present invention can be innoculated with, infectedwith, or otherwise exposed to virus by any method known in the art. Insome embodiments, an animal host may be innoculated with, infected with,or exposed to virus intranasally.

In some embodiments, a suitable animal host may have a similardistribution of umbrella vs. cone topology glycans and/or α2-6 glycansvs. α 2-3 glycans to the distribution found in the human respiratorytract. For example, it is contemplated that a ferret as an animal hostmay be more representative than a mouse when used as model of diseasecaused by influenza viruses in humans (Tumpey et al., 2007, Science 315;655-59; incorporated herein by reference). Without wishing to be boundany theories, the present invention encompasses the idea that ferretsmay have a more similar distribution of glycans in the respiratory tractto those in the human respiratory tract than mouse does to human. Naïveand/or innoculated animals may be used for any of a variety of studies.For example, such animal models may be used for virus transmissionstudies as in known in the art. It is contemplated that the use offerrets in virus transmission studies may serve as a reliable predictorfor virus transmission in humans. For example, air transmission of viralinfluenza from innoculated animals (e.g., ferrets) to naïve animals isknown in the art (Tumpey et al., 2007, Science 315; 655-59; incorporatedherein by reference). Virus transmission studies may be used to testbinding agent polypeptides in accordance with the invention (e.g., HApolypeptides). For example, binding agents in accordance with theinvention may be administered to a suitable animal host before, duringor after virus transmission studies in order to determine the efficacyof said binding agent in blocking virus binding and/or infectivity inthe animal host. Using information gathered from virus transmissionstudies in an animal host, one may predict the efficacy of a bindingagent in blocking virus binding and/or infectivity in a human host.

Pharmaceutical Compositions

In some embodiments, the present invention provides for pharmaceuticalcompositions including binding agents in accordance with the invention(e.g., HA polypeptides, LSBAs, UTBAs, UTBSAs, etc.) and/or relatedentities. For example, in some embodiments, binding agent polypeptide(s)(e.g., HA polypeptides), nucleic acids encoding such polypeptides,characteristic or biologically active fragments of such polypeptides ornucleic acids, antibodies that bind to and/or compete with suchpolypeptides or fragments, small molecules that interact with or competewith such polypeptides or with glycans that bind to them, etc. areincluded in pharmaceutical compositions in accordance with theinvention.

The invention encompasses treatment of influenza infections byadministration of such pharmaceutical compositions in accordance withthe invention. In some embodiments, pharmaceutical compositions inaccordance with the invention are administered to a subject sufferingfrom or susceptible to an influenza infection. In some embodiments, asubject is considered to be suffering from an influenza infection in thesubject is displaying one or more symptoms commonly associated withinfluenza infection. In some embodiments, the subject is known orbelieved to have been exposed to the influenza virus. In someembodiments, a subject is considered to be susceptible to an influenzainfection if the subject is known or believed to have been exposed tothe influenza virus. In some embodiments, a subject is known or believedto have been exposed to the influenza virus if the subject has been incontact with other individuals known or suspected to have been infectedwith the influenza virus and/or if the subject is or has been present ina location in which influenza infection is known or thought to beprevalent.

In some embodiments, subjects suffering from or susceptible to influenzainfection are tested for antibodies to binding agents in accordance withthe invention prior to, during, or after administration ofpharmaceutical compositions in accordance with the invention. In someembodiments, subjects having such antibodies are not administeredpharmaceutical compositions comprising binding agents in accordance withthe invention. In some embodiments, an appropriate dose ofpharmaceutical composition and/or binding agent is selected based ondetection (or lack thereof) of such antibodies.

In some embodiments, selection of a particular subject for treatment,particular binding agent or composition for administration, and/orparticular dose or regimen for administration, is memorialized, forexample in a written, printed, or electronic storage form.

Compositions in accordance with the invention may be administered priorto or after development of one or more symptoms of influenza infection.

The invention encompasses treatment of influenza infections byadministration of compounds described herein. In some embodiments,treatment of influenza infections according to the present invention isaccomplished by administration of a vaccine. To date, althoughsignificant accomplishments have been made in the development ofinfluenza vaccines, there is room for further improvement. The presentinvention provides vaccines comprising binding agents in accordance withthe invention (e.g., HA polypeptides, LSBAs, UTBAs, UTBSAs, etc.), andparticularly comprising binding agents that bind to umbrella glycans(e.g., α2-6 linked umbrella glycans such as, for example, long α2-6sialylated glycans).

To give but one example, attempts to generate vaccines specific for theH5N1 strain in humans have generally not been successful due, at leastin part, to low immunogenicity of H5 HAs. In one study, a vaccinedirected at the H5N1 strain was shown to yield antibody titers of 1:40,which is not a titer high enough to guarantee protection from infection.Furthermore, the dosage required to generate even a modest 1:40 antibodytiter (two doses of 90 μg of purified killed virus or antigen) was12-times that normally used in the case of the common seasonal influenzavirus vaccine (Treanor et al., 2006, N Eng J Med, 354:1343; incorporatedherein by reference). Other studies have similarly shown that current H5vaccines are not highly immunogenic (Bresson et al., 2006, Lancet,367:1657; incorporated herein by reference). In some embodiments,vaccines in accordance with the invention are formulated utilizing oneor more strategies (see, for example, Enserink, 2005, Science, 309:996;incorporated herein by reference) intended to allow use of lower dose ofH5 HA protein, and/or to achieve higher immunogenicity. For example, insome embodiments, multivalency is improved (e.g., via use ofdendrimers); in some embodiments, one or more adjuvants is utilized,etc.

In some embodiments, the present invention provides for vaccines and theadministration of these vaccines to a human subject. In someembodiments, vaccines are compositions comprising one or more of thefollowing: (1) inactivated virus, (2) live attenuated influenza virus,for example, replication-defective virus, (3) binding agent inaccordance with the invention (e.g., HA polypeptides, LSBAs, UTBAs,UTBSAs, etc.), (4) nucleic acid encoding binding agent polypeptide(e.g., HA polypeptide) or characteristic or biologically active portionthereof, (5) DNA vector that encodes binding agent polypeptide inaccordance with the invention (e.g., HA polypeptide) or characteristicor biologically active portion thereof, and/or (6) expression system,for example, cells expressing one or more influenza proteins to be usedas antigens.

Thus, in some embodiments, the present invention provides inactivatedflu vaccines. In some embodiments, inactivated flu vaccines comprise oneof three types of antigen preparation: inactivated whole virus,sub-virions where purified virus particles are disrupted with detergentsor other reagents to solubilize the lipid envelope (“split” vaccine) orpurified HA polypeptide (“subunit” vaccine). In some embodiments, viruscan be inactivated by treatment with formaldehyde, beta-propiolactone,ether, ether with detergent (such as) TWEEN-80®), cetyl trimethylammonium bromide (CTAB) and Triton N101, sodium deoxycholate andtri(n-butyl) phosphate. Inactivation can occur after or prior toclarification of allantoic fluid (from virus produced in eggs); thevirions are isolated and purified by centrifugation (Nicholson et al.,eds., 1998, Textbook of Influenza, Blackwell Science, Malden, Mass.;incorporated herein by reference). To assess the potency of the vaccine,the single radial immunodiffusion (SRD) test can be used (Schild et al.,1975, Bull. World Health Organ., 52:43-50 & 223-31; Mostow et al., 1975,J. Clin. Microbiol., 2:531; both of which are incorporated herein byreference).

The present invention also provides live, attenuated flu vaccines, andmethods for attenuation are well known in the art. In some embodiments,attenuation is achieved through the use of reverse genetics, such assite-directed mutagenesis.

In some embodiments, influenza virus for use in vaccines is grown ineggs, for example, in embryonated hen eggs, in which case the harvestedmaterial is allantoic fluid. Alternatively or additionally, influenzavirus may be derived from any method using tissue culture to grow thevirus. Suitable cell substrates for growing the virus include, forexample, dog kidney cells such as MDCK or cells from a clone of MDCK,MDCK-like cells, monkey kidney cells such as AGMK cells including Verocells, cultured epithelial cells as continuous cell lines, 293T cells,BK-21 cells, CV-1 cells, or any other mammalian cell type suitable forthe production of influenza virus (including upper airway epithelialcells) for vaccine purposes, readily available from commercial sources(e.g., ATCC, Rockville, Md.). Suitable cell substrates also includehuman cells such as MRC-5 cells. Suitable cell substrates are notlimited to cell lines; for example primary cells such as chicken embryofibroblasts are also included.

In some embodiments, vaccines in accordance with the invention furthercomprise one or more adjuvants. For example, aluminum salts (Baylor etal., 2002, Vaccine, 20:S18; incorporated herein by reference) andmonophosphoryl lipid A (MPL; Ribi et al., 1986, Immunology andImmunopharmacology of Bacterial Endotoxins, Plenum Publ. Corp., NY, p.407; incorporated herein by reference) can be used as adjuvants in humanvaccines. Alternatively or additionally, new compounds are currentlybeing tested as adjuvants in human vaccines, such as MF59 (Chiron Corp.,available through the world wide web atchiron.com/investors/pressreleases/2005/051028, CPG 7909 (Cooper et al.,2004, Vaccine, 22:3136; incorporated herein by reference), and saponins,such as QS21 (Ghochikyan et al., 2006, Vaccine, 24:2275; incorporatedherein by reference).

Additionally, some adjuvants are known in the art to enhance theimmunogenicity of influenza vaccines, such aspoly[di(carboxylatophenoxy)phosphazene] (PCCP; Payne et al., 1998,Vaccine, 16:92; incorporated herein by reference), interferon-γ (Cao etal., 1992, Vaccine, 10:238; incorporated herein by reference), blockcopolymer P1205 (CRL1005; Katz et al., 2000, Vaccine, 18:2177;incorporated herein by reference), interleukin-2 (IL-2; Mbwuike et al.,1990, Vaccine, 8:347; incorporated herein by reference), and polymethylmethacrylate (PMMA; Kreuter et al., 1981, J. Pharm. Sci., 70:367;incorporated herein by reference).

In some embodiments, compositions in accordance with the invention,e.g., compositions of binding agents, do not include adjuvants (e.g.,provided compositions are essentially free of adjuvants). In someembodiments, compositions in accordance with the invention do notinclude an alum adjuvant (e.g., provided compositions are essentiallyfree of alum).

In addition to vaccines, the present invention provides othertherapeutic compositions useful in the treatment of viral infections. Insome embodiments, treatment is accomplished by administration of anagent that interferes with expression or activity of an HA polypeptide.

In some embodiments, the present invention provides pharmaceuticalcompositions comprising antibodies or other agents related to providedpolypeptides. For example, the invention provides compositionscontaining antibodies recognize virus particles containing a particularHA polypeptide (e.g., an HA polypeptide that binds to umbrella glycans),nucleic acids (such as nucleic acid sequences complementary to HAsequences, which can be used for RNAi), glycans that compete for bindingto HA receptors, small molecules or glycomimetics that compete theglycan-HA polypeptide interaction, or any combination thereof. In someembodiments, collections of different agents, having diverse structuresare utilized. In some embodiments, therapeutic compositions comprise oneor more multivalent agents. In some embodiments, treatment comprisesurgent administration shortly after exposure or suspicion of exposure.

In some embodiments, any of the vaccines described herein offer broadcross-protection against different varieties of influenza viruses. Forexample, in some embodiments, vaccines described herein offercross-protection against both avian and human-adapted H5 viruses. Insome embodiments, any of the vaccines described herein offercross-protection against any H5 influenza virus strain or variant. Insome embodiments, any of the vaccines described herein offercross-protection against any H2 influenza virus strain or variant. Insome embodiments, any of the vaccines described herein offercross-protection against any H1, H2, H3, H4, H5, H6, H7, H8, H9, H10,H11, H12, H13, H14, H15, or H16 influenza virus strain or variant.

In general, a pharmaceutical composition will include a therapeuticagent in addition to one or more inactive agents such as a sterile,biocompatible carrier including, but not limited to, sterile water,saline, buffered saline, or dextrose solution. Alternatively oradditionally, the composition can contain any of a variety of additives,such as stabilizers, buffers, excipients (e.g., sugars, amino acids,etc.), or preservatives.

In some embodiments, the therapeutic agent present in a pharmaceuticalcomposition in accordance with the invention will consist of one or morebinding agents as described herein. In some embodiments, apharmaceutical composition in accordance with the invention contains abinding agent (e.g., an HA polypeptide, LSBA, UTBA, UTSBA, etc.) thatbinds to umbrella topology glycans (and/or to umbrella topology glycanmimics). In some such embodiments, the composition in accordance withthe invention is substantially free of related agents (e.g., of other HApolypeptides, etc.) that do not bind to umbrella-topology glycans. Insome such embodiments, pharmaceutical compositions in accordance withthe invention contains not more than 50%, 40%, 30%, 20%, 10%, 5%, or 1%of an agent that binds to HA receptor glycans other than umbrellatopology glycans.

In some embodiments, a pharmaceutical composition will include atherapeutic agent that is encapsulated, trapped, or bound within a lipidvesicle, a bioavailable and/or biocompatible and/or biodegradablematrix, or other microparticle.

In some embodiments, a provided pharmaceutical composition will includea binding agent (e.g., an HA polypeptide, LSBA, UTBA, UTSBA, etc.) thatis not aggregated. For example, in some embodiments, less than 1%, 2%,5%, 10%, 20%, or 30%, by dry weight or number, of the binding agent ispresent in an aggregate.

In some embodiments, a provided pharmaceutical composition will includea binding agent (e.g., an HA polypeptide, LSBA, UTBA, UTSBA, etc.) thatis not denatured. For example, in some embodiments, less than 1%, 2%,5%, 10%, 20%, or 30%, by dry weight or number, of the UTSBA administeredis denatured.

In some embodiments, a provided pharmaceutical composition will includea binding agent (e.g., an HA polypeptide, LSBA, UTBA, UTSBA, etc.) thatis not inactive. For example, in some embodiments, less than 1%, 2%, 5%,10%, 20%, or 30%, by dry weight or number, of the UTSBA administered isinactive.

In some embodiments, pharmaceutical compositions in accordance with theinvention are formulated to reduce immunogenicity of provided bindingagents. For example, in some embodiments, a provided binding agent isassociated with (e.g., bound to) an agent, such as polyethylene glycoland/or carboxymethyl cellulose, that masks its immunogenicity. In someembodiments, a provided binding agent has additional glycosylation thatreduces immunogenicity.

Pharmaceutical compositions of the present invention may be administeredeither alone or in combination with one or more other therapeutic agentsincluding, but not limited to, vaccines and/or antibodies. By “incombination with,” it is not intended to imply that the agents must beadministered at the same time or formulated for delivery together,although these methods of delivery are within the scope of theinvention. In general, each agent will be administered at a dose and ona time schedule determined for that agent. Additionally, the inventionencompasses the delivery of pharmaceutical compositions in accordancewith the invention in combination with agents that may improve theirbioavailability, reduce or modify their metabolism, inhibit theirexcretion, or modify their distribution within the body. Although thepharmaceutical compositions of the present invention can be used fortreatment of any subject (e.g., any animal) in need thereof, they aremost preferably used in the treatment of humans. In some embodiments,pharmaceutical compositions in accordance with the invention and/orbinding agents are administered in combination with one or more of ananti-viral agent (e.g., Oseltamivir [TAMIFLU®], Zanamavir [RELEZA®],etc.) and/or a sialidase.

Pharmaceutical compositions of the present invention can be administeredby a variety of routes, including oral, intravenous, intramuscular,intra-arterial, subcutaneous, intraventricular, transdermal,interdermal, rectal, intravaginal, intraperitoneal, topical (as bypowders, ointments, creams, or drops), mucosal, nasal, buccal, enteral,sublingual; by intratracheal instillation, bronchial instillation,and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.In general the most appropriate route of administration will depend upona variety of factors including the nature of the agent (e.g., itsstability in the environment of the gastrointestinal tract), thecondition of the patient (e.g., whether the patient is able to tolerateoral administration), etc.

At present the oral or nasal spray or aerosol route (e.g., byinhalation) is most commonly used to deliver therapeutic agents directlyto the lungs and respiratory system. However, the invention encompassesthe delivery of the pharmaceutical composition in accordance with theinvention by any appropriate route taking into consideration likelyadvances in the sciences of drug delivery.

In some embodiments, preparations for inhaled or aerosol deliverycomprise a plurality of particles. In some embodiments, suchpreparations have a mean particle size of about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, or about 13 microns. In some embodiments, preparations forinhaled or aerosol delivery are formulated as a dry powder. In someembodiments, preparations for inhaled or aerosol delivery are formulatedas a wet powder, for example through inclusion of a wetting agent. Insome embodiments, the wetting agent is selected from the groupconsisting of water, saline, or other liquid of physiological pH.

In some embodiments, compositions in accordance with the invention areadministered as drops to the nasal or buccal cavity. In someembodiments, a dose may comprise a plurality of drops (e.g., 1-100,1-50, 1-20, 1-10, 1-5, etc.)

In some embodiments, compositions in accordance with the invention areadministered using a device that delivers a metered dosage ofcomposition (e.g., of binding agent).

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499, 5,190,521, 5,328,483, 5,527,288,4,270,537, 5,015,235, 5,141,496, 5,417,662 (all of which areincorporated herein by reference). Intradermal compositions may also beadministered by devices which limit the effective penetration length ofa needle into the skin, such as those described in WO99/34850,incorporated herein by reference, and functional equivalents thereof.Also suitable are jet injection devices which deliver liquid vaccines tothe dermis via a liquid jet injector or via a needle which pierces thestratum corneum and produces a jet which reaches the dermis. Jetinjection devices are described for example in U.S. Pat. Nos. 5,480,381,5,599,302, 5,334,144, 5,993,412, 5,649,912, 5,569,189, 5,704,911,5,383,851, 5,893,397, 5,466,220, 5,339,163, 5,312,335, 5,503,627,5,064,413, 5,520,639, 4,596,556, 4,790,824, 4,941,880, 4,940,460, WO97/37705, and WO 97/13537 (all of which are incorporated herein byreference). Also suitable are ballistic powder/particle delivery deviceswhich use compressed gas to accelerate vaccine in powder form throughthe outer layers of the skin to the dermis. Additionally, conventionalsyringes may be used in the classical mantoux method of intradermaladministration.

General considerations in the formulation and manufacture ofpharmaceutical agents may be found, for example, in Remington'sPharmaceutical Sciences, 19^(th) ed., Mack Publishing Co., Easton, Pa.,1995; incorporated herein by reference.

Pharmaceutical compositions in accordance with the invention may beadministered in any dose appropriate to achieve a desired outcome. Insome embodiments, the desired outcome is reduction in intensity,severity, and/or frequency, and/or delay of onset of one or moresymptoms of influenza infection.

In some embodiments, pharmaceutical compositions in accordance with theinvention are formulated to administer a dose of binding agent effectiveto compete with influenza HA for binding to umbrella topology glycans.In some embodiments, such binding by influenza HA is reduced afteradministration of one or more doses of a composition in accordance withthe invention as compared with its level absent such administration. Insome embodiments, pharmaceutical compositions in accordance with theinvention are formulated to administer a dose of binding agent effectiveto saturate at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or more HA binding sites (e.g., HA bindingsites containing umbrella topology glycans) present in the subject(e.g., in the respiratory tract of the subject) receiving thecomposition.

In some embodiments, pharmaceutical compositions in accordance with theinvention are formulated to deliver a unit dose of binding agent withinthe range of 0.0001 to 1000 mg/kg.

In some embodiments, pharmaceutical compositions in accordance with theinvention are administered in multiple doses. In some embodiments,pharmaceutical compositions in accordance with the invention areadministered in multiple doses/day. In some embodiments, pharmaceuticalcompositions in accordance with the invention are administered accordingto a continuous dosing regimen, such that the subject does not undergoperiods of less than therapeutic dosing interposed between periods oftherapeutic dosing. In some embodiments, pharmaceutical compositions inaccordance with the invention are administered according to anintermittent dosing regimen, such that the subject undergoes at leastone period of less than therapeutic dosing interposed between twoperiods of therapeutic dosing.

Diagnostics/Kits

The present invention provides kits for detecting binding agents (e.g.,HA polypeptides, LSBAs, UTBAs, UTSBAs, etc), and particular fordetecting binding agents with particular glycan binding characteristics(e.g., binding to umbrella glycans, to α2-6 sialylated glycans, to longα2-6 sialylated glycans, etc.) in pathological samples, including, butnot limited to, blood, serum/plasma, peripheral blood mononuclearcells/peripheral blood lymphocytes (PBMC/PBL), sputum, urine, feces,throat swabs, dermal lesion swabs, cerebrospinal fluids, cervicalsmears, pus samples, food matrices, and tissues from various parts ofthe body such as brain, spleen, and liver. The present invention alsoprovides kits for detecting binding agents (e.g., HA polypeptides,LSBAs, UTBAs, UTSBAs, etc) of interest in environmental samples,including, but not limited to, soil, water, and flora. Other samplesthat have not been listed may also be applicable.

In some embodiments, the present invention provides kits for detectingHA polypeptides as described herein whether or not such polypeptides arebinding agents.

In some embodiments, kits in accordance with the invention may includeone or more agents that specifically detect binding agents (e.g., HApolypeptides, LSBAs, UTBAs, UTSBAs, etc) with particular glycan bindingcharacteristics. Such detecting agents may include, for example,antibodies that specifically recognize certain binding agents (e.g.,binding agents that bind to umbrella glycans and/or to α2-6 sialylatedglycans and/or to long α2-6 sialylated glycans), which can be used tospecifically detect such binding agents by ELISA, immunofluorescence,and/or immunoblotting.

Antibodies that bind to HA polypeptides can also be used in virusneutralization tests, in which a sample is treated with antibodyspecific to HA polypeptides of interest, and tested for its ability toinfect cultured cells relative to untreated sample. If the virus in thatsample contains such HA polypeptides, the antibody will neutralize thevirus and prevent it from infecting the cultured cells. Alternatively oradditionally, such antibodies can also be used in HA-inhibition tests,in which the HA protein is isolated from a given sample, treated withantibody specific to a particular HA polypeptide or set of HApolypeptides, and tested for its ability to agglutinate erythrocytesrelative to untreated sample. If the virus in the sample contains suchan HA polypeptide, the antibody will neutralize the activity of the HApolypeptide and prevent it from agglutinating erythrocytes (Harlow &Lane, 1988, Antibodies: A Laboratory Manual, CSHL Press; availablethrough the world wide web atwho.int/csr/resources/publications/influenza/WHO_CDS_CSR_NCS_2002_5/en/indexand who.int/csr/disease/avian_influenza/guidelines/labtests/en/index).In some embodiments, such agents may include nucleic acids thatspecifically bind to nucleotides that encode particular HA polypeptidesand that can be used to specifically detect such HA polypeptides byRT-PCR or in situ hybridization (available through the world wide web atwho.int/csr/resources/publications/influenza/WHO_CDS_CSR_NCS_2002_5/en/indexand who.int/csr/disease/avian_influenza/guidelines/labtests/en/index).In some embodiments, nucleic acids which have been isolated from asample are amplified prior to detection. In some embodiments, diagnosticreagents can be detectably labeled.

The present invention also provides kits containing reagents accordingto the invention for the generation of influenza viruses and vaccines.Contents of the kits include, but are not limited to, expressionplasmids containing HA nucleotides (or characteristic or biologicallyactive portions) encoding HA polypeptides of interest (or characteristicor biologically active portions). Alternatively or additionally, kitsmay contain expression plasmids that express HA polypeptides of interest(or characteristic or biologically active portions). Expression plasmidscontaining no virus genes may also be included so that users are capableof incorporating HA nucleotides from any influenza virus of interest.Mammalian cell lines may also be included with the kits, including butnot limited to, Vero and MDCK cell lines. In some embodiments,diagnostic reagents can be detectably labeled.

In some embodiments, kits for use in accordance with the presentinvention may include, a reference sample, instructions for processingsamples, performing the test, instructions for interpreting the results,buffers and/or other reagents necessary for performing the test. In someembodiments the kit can comprise a panel of antibodies.

In some embodiments of the present invention, glycan arrays, asdiscussed above, may be utilized as diagnostics and/or kits.

In some embodiments, glycan arrays and/or kits in accordance with theinvention are used to perform dose response studies to assess binding ofHA polypeptides to umbrella glycans at multiple doses (e.g., asdescribed herein). Such studies give particularly valuable insight intothe binding characteristics of tested HA polypeptides, and areparticularly useful to assess specific binding. Dose response bindingstudies of this type find many useful applications. To give but oneexample, they can be helpful in tracking the evolution of bindingcharacteristics in a related series of HA polypeptide variants, whetherthe series is generated through natural evolution, intentionalengineering, or a combination of the two.

In some embodiments, glycan arrays and/or kits in accordance with theinvention are used to induce, identify, and/or select binding agents(e.g., HA polypeptides, and/or HA polypeptide variants) having desiredbinding characteristics. For instance, in some embodiments, glycanarrays and/or kits in accordance with the invention are used to exertevolutionary (e.g., screening and/or selection) pressure on a populationof polypeptide binding agents (e.g., HA polypeptides).

The present invention provides kits for administration of pharmaceuticalcompositions in accordance with the invention. For example, in someembodiments, the invention provides a kit comprising at least one doseof a binding agent. In some embodiments, the invention provides a kitcomprising an initial unit dose and a subsequent unit dose of a bindingagent. In some such embodiments, the initial unit dose is greater thanthe subsequent unit dose or wherein the two doses are equal.

In some embodiments, kits in accordance with the invention (particularlythose for administration of pharmaceutical compositions in accordancewith the invention) comprise at least one component of a deliverydevice, e.g., an inhaler. In some such embodiments, the inventionprovides a kit comprising at least one component of a delivery device,e.g., an inhaler and a dose of a binding agent.

In some embodiments, provided kits comprise instructions for use.

EXEMPLIFICATION Example 1 Identification of Molecular Determinants ofBroad Spectrum Human Binding HA Polypeptides

Introduction

The H5N1 avian influenza virus (“bird flu” or “avian flu”) is a highlyinfectious and deadly pathogen. Since 1996, several H5N1 outbreaks haveoccurred across three continents killing millions of poultry. Since itsemergence, the virus has also shown potential to infect humans with amortality rate exceeding 60%. Humans have virtually no immunity to theH5N1 virus, but this virus has not yet adapted to the human host to beable to efficiently infect and transmit between humans. The virus wouldacquire mutations that allow it to gain a foothold in the humanpopulation.

Hemagglutinin (HA), the surface glycoprotein of influenza A virus, isresponsible for initiating viral entry into the host cell. HA binds tosialylated glycan receptors (complex glycans terminated by α2→3 orα2→6—linked sialic acid). The H5N1 HA preferentially binds to glycanreceptors terminated by α2→3 linked sialic acid. The present inventorshave demonstrated that the glycan receptors in the human host forhuman-adapted influenza A viruses are α2→6 sialylated glycans that adopta characteristic umbrella-like topology (referred to henceforth as humanreceptors) in the glycan receptor-binding site (RBS) of HA(Chandrasekharan et al., 2008, Nat Biotech, 26:107; incorporated hereinby reference). The present inventors have also shown that high affinitybinding to these human glycan receptors is a characteristic of humanadapted HA and correlates with the efficient airborne transmissibilityof the human-adapted H1N1 and H3N2 viruses (Chandrasekharan et al.,2008, Nat Biotech, 26:107; Srinivasan et al., 2008, PNAS, 105: 2800;both of which are incorporated herein by reference. In the presentstudy, the present inventors have provided an H5N1 virus in which HA hassequence substitutions in the RBS that allow it to bind with highaffinity to human receptors.

Experimental Design

Influenza HA is a homotrimeric protein, wherein a monomer contains 552amino acids. Each monomer is composed of two disulphide-linked moieties,HA1 and HA2. HA1 comprises the glycan-receptor binding site (RBS),whereas HA2 is involved in the fusion of the viral and cellularmembranes. The RBS pocket involves HA positions 95, 131, 133, 136, 137,138, 145, 153, 155, 156, 158, 159, 183, 186, 187, 189, 190, 192, 193,194, 195, 196, 219, 222, 224, 225, 226, 227, 228 (H3 numbering used).

The present inventors have conducted a detailed analysis of molecularcontacts between H5N1 and a representative human receptor and havecompared these contacts with those between the human-adapted pandemicH1N1, H2N2 and H3N2 HAs and human receptors. Through this approach, thepresent inventors have defined strategies for generating mutant forms ofH5N1 HA. The mutations Q226L and G228S (or LS) have been introducedacross several genetic clades of H5N1 HA. These mutations are based onthe characteristic amino acid substitutions at the 226 and 228 positionsof H2N2 and H3N2 HAs that have led to their human adaptation,respectively.

These mutations have been introduced in the context of the RBS ofdifferent genetic clades of H5N1 HA (FIG. 13). Previous studies (e.g.,Stevens et al., 2008, J. Mol. Biol., 381:1382-94; and Stevens et al.,2006, Science, 312:404; both of which are incorporated herein byreference) have analyzed both recombinant HA and whole virusescomprising the LS mutations in different H5N1 strains on glycan arrayplatforms and have shown that some of these strains have acquiredbinding to α2→6 sialylated glycans (FIG. 14). However, the goal of thesestudies was to screen for binding at high protein concentration or viraltiters to determine how many α2→3 or α2→6 glycans showed bindingsignals.

In contrast, the present inventors performed a dose-dependent analysison recombinant HAs carrying the LS mutation in the RBS from several H5N1strains (FIG. 15). The present data demonstrate that none of thesemutants showed the characteristic high binding affinity to humanreceptors that is shared by human-adapted HAs. The present results alsocorrelated with the inefficient transmission of H5N1 viruses carryingjust the LS mutation in the RBS. However, none of these mutants showedhigh affinity human receptor binding. Therefore additional strategieswere needed to define mutant forms of H5N1 HA.

Mutations that Abrogate Glycosylation

Thus, the present inventors introduced 5 amino acid substitutions at128, 133, 145, 159, 193 (bold and underlined residues below) into theViet_1203_04_c1 (A/Viet Nam/1203/04 which is a clade 1 strain) HA inorder to make its RBS similar to that of the more recent genetic clades(which includes strains such as A/Indonesia/5/05).

The present inventors recognized that glycosylation at amino acidposition N158 might interfere with the umbrella-like topology of humanreceptor in the RBS of H5N1 HA. Thus, the inventors' first strategyinvolved generating mutant forms of H5N1 HA that contained Q228L and/orG228S substitutions and an additional T160A mutation that abrogatesglycosylation at N158.

This glycosylation site is not conserved across all genetic clades. Ifthe absence of glycosylation at N158 improves human receptor binding ofH5N1 HA with LS mutations, then the LS mutations alone might suffice toachieve human adaptation of viruses belonging to genetic clades thatnaturally lack this glycosylation site (c2.2, c2.2.1, etc.; see FIG.13). The template sequence for these clades was chosen asEgypt_2876-N3_06_c2.2 (A/Egypt/2786-N3/06 belonging to clade 2.2). Thepresent inventors have also defined mutant forms that carry the Q226Lmutation alone (in the context of deglycosylation at 158 position) tocheck the extent of improvement of the human receptor binding affinity,given that in H2 HA, the Q226L mutation is sufficient to substantiallyimprove the affinity.

Dose-dependent analysis of the above mutants (FIG. 16) showed thatremoving the glycosylation at Asn-158 by mutating Thr-160→Ala in thecontext of LS mutation substantially improves binding to humanreceptors. This was observed for both the T160A/Q226L/G228S mutant onViet1203_04 D as well as LS mutation on Egypt_2876-N3_06_c2.2 HA. Thepresent inventors further demonstrated that the Q226L mutation alone(without G228S) is not sufficient to provide the high humanreceptor-binding affinity in the context of the removal of glycosylationat N158. The present invention, however, encompasses the recognitionthat a mutation at Q226 (e.g., Q226L) alone might suffice to provide thehigh human receptor-binding affinity in the context of the removal ofglycosylation at N158 in the context of some particular virus strains.The present invention also encompasses the recognition that a mutationat G228 (e.g., G228S) alone might suffice to provide the high humanreceptor-binding affinity in the context of the removal of glycosylationat N158 in the context of some particular virus strains. The presentinvention also encompasses the recognition that mutations at both Q226and G228 (e.g., Q226L and G228S) might be required to provide the highhuman receptor-binding affinity in the context of the removal ofglycosylation at N158 in the context of some particular virus strains.

In addition, the inventors discovered that binding of the above mutantsto avian receptors (particularly to 3′SLN-LN-LN and 3′SLN-LN) was alsoquite high, which is not typical of human adapted HAs. For example, evenin the case of the prototypic pandemic H2N2 HA (A/Albany/6/58) whichshowed both human and avian receptor binding, the human receptor bindingaffinity was orders of magnitude higher than that of avianreceptor-binding affinity.

Amino Acid Deletion in the Loop Region

The present inventors have designed mutant forms of H5N1 HA such thatthe molecular contacts between its RBS and human receptor closely mimicsthat of contacts between human-adapted H2N2 HAs and human receptors.Based on the inventors' previous work in understanding the molecularcomposition of the RBS of H2N2 HA and how it governs glycan receptorspecificity, the inventors first performed a detailed molecularcomparison of the RBS of H5N1 HA with that of a prototypic human-adaptedH2N2 HA from a 1957-58 pandemic strain (A/Albany/6//58 or Alb6_58) (FIG.17). In order to minimize the differences in the molecular compositionof RBS between H5 HA and Alb6_58 HA, the inventors generated a mutant onthe Viet1203_04 D template (“Viet1203_04_D_H2RBS”; SEQ ID NO: 61) thatcomprised 13 amino acid substitutions at 131, 132, 133, 135, 137, 155,188, 192, 193, 221, 226, 227, and 228, and a deletion at 130. Thepresent study, for the very first time, focuses on design and testing ofmutations in the H5N1 HA RBS that represent a combination of deletionand substitutions based on a comprehensive comparison between H2 and H5HA. The deletion was made at position 130. Although H2N2 HA lacksglycosylation at 158 position, the N158 glycosylation site (ofViet1203_04_D) in the mutant HA was retained. Thus, the presentinvention encompasses the recognition that mutations that abrogateglycosylation at the 158 site in addition to the aforementionedmutations might enhance human binding even further. The presentinventors also designed another version of this mutant(“Viet1203_04_D_H2RBSmin”; SEQ ID NO: 63) with fewer mutations based onconservative substitutions.

The present inventors also searched for HA sequences from other H5N1strains which might naturally be closer to Alb6_58 H2N2 HA in terms ofmolecular composition of the RBS. The inventors identified two sets ofexemplary templates: (1) with the switch in charge properties ofpositions 192 and 193 (A/chicken/Vietnam/NCVD-093/2008 or ckViet_08),and (2) with a deletion in the 130 loop (A/chicken/Egypt/R2/2007 orckEgy_07). Additional mutant forms with fewer mutations in theabove-mentioned positions on these new H5N1 HA templates were designedto make the H5N1 HA RBS mimic that of human-adapted H2N2 HA RBS.

Mutant human-receptor binding affinity using dose-dependent glycan arraywere tested. The Viet1203_04 D_H2RBS mutant showed highly specific highaffinity binding to human receptors that is characteristic of humanadapted H1N1 and H2N2 HAs (FIG. 19). The present invention encompassesthe recognition that additional mutations can be designed to understandthe relationship between (1) the 130 loop composition and deletion, (2)switch in the charged residues at the 192 and 193 positions, and (3)glycosylation at 158 position and how this relationship governs thehuman receptor binding affinity of the mutant H5N1 HAs.

Exemplary Sequences of H5N1 Templates Used and Exemplary MutantPolypeptides Designed in Accordance with the Above Principles and thePrinciples Set Forth in Example 2

An alignment of exemplary H5N1 templates and exemplary mutant H5 HApolypeptides designed in accordance with the above principles and theprinciples set forth in Example 2 are presented in FIG. 18 and below:

Viet1203_04_D: Bold, Underlined Residues Denote Substitution Sites

(SEQ ID NO: 60) MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWS D HEAS S GVSSA CPYQGK PSFFRNVVWLIKKN N TYPTIKRSYNNTNQEDLLVLWGIHHPNDA AEQT RLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVA GLSLWMCSNGSLQCRICI

In the following sequences, italics and highlighting denote the regionwhere the mutations are made, and boldface and underlining indicate theresidues that were mutated.

Viet1203_04_D_H2RBS: (SEQ ID NO: 61)MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFE

FESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICIViet1203_04_D_H2RBS_N158deglyc (SEQ ID NO: 62)MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFE

FESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICIViet1203_04_D_H2RBSmin): Deletion + 7 mutations (SEQ ID NO: 63)MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFE

FESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICIViet1203_04_D (T160A/Q226L/G228S) mutant: (SEQ ID NO: 64)MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFE

FESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICIEgypt_2876-N3_06_c2.2: Lack of glycosylation at 158 is indicated bylarger font (SEQ ID NO: 65)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFE

WGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICIEgypt_2876-N3_06_c2.2 HA with LS mutation: Lack of glycosylation at158 is indicated by law font (SEQ ID NO: 66)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFE

WGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNG L S S RMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICIEgypt_2876-N3_06_c2.2 HA with a single Q226L mutation: Lack ofglycosylation at 158 is indicated by larger font (SEQ ID NO: 67)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFE

WGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNG L SGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICIViet1203_04_D with T160A/Q226L double mutation: (SEQ ID NO: 68)MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGKPSFFRNVVWLIKKNN A YPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPRIATRSKVNG L SGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICIckViet_08: WT HA that already possesses the Lys192 and Met193 chargecombination as observed in H2N2 (indicated by larger font)(SEQ ID NO: 69)MEKIVLLLAIIGLVKSDQICVGYHANNSTEQVDTIMEKNITVTHAQDILEKTHNGKLCNLNGVKPLILKDCSVAGWLLGNPMCDEFLNVSEWSYIVEKASPANGLCYPGDFNDYEELKHLLSRINHFEKIKIIPKSYWSNHETSLGVSSACSYLENPSFFRNVVWLTKKNNTYPPIKVNYTNANQKDLLVLWGIHHPNNEAEQKMIYQNLNTYVSVGTSTLNQRLVPKIATRSKVNGQSGRMDFFWTILKPNDTINFDSNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNAPQIEGRRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGITNKINSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYEKVRLQLRDNAKELGNGCFEFYHKCDNECMESVKNGTYDYPQYSEEARLNREEISGVKLESIVTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICIckViet_08_H2RBS: Deletion + 6 mutations (SEQ ID NO: 70)MEKIVLLLAIIGLVKSDQICVGYHANNSTEQVDTIMEKNITVTHAQDILEKTHNGKLCNLNGVKPLILKDCSVAGWLLGNPMCDEFLNVSEWSYIVEKASPANGLCYPGDFNDYEELKHLLSRINHFE

NFDSNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNAPQIEGRRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGITNKINSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYEKVRLQLRDNAKELGNGCFEFYHKCDNECMESVKNGTYDYPQYSEEARLNREEISGVKLESIVTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICIckViet_08_H2RBS: Deletion + 5 mutations (SEQ ID NO: 71)MEKIVLLLAIIGLVKSDQICVGYHANNSTEQVDTIMEKNITVTHAQDILEKTHNGKLCNLNGVKPLILKDCSVAGWLLGNPMCDEFLNVSEWSYIVEKASPANGLCYPGDFNDYEELKHLLSRINHFE

NFDSNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNAPQIEGRRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGITNKINSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYEKVRLQLRDNAKELGNGCFEFYHKCDNECMESVKNGTYDYPQYSEEARLNREEISGVKLESIVTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICIckViet_08_H2RBSmin: Deletion + 4 mutations (SEQ ID NO: 72)MEKIVLLLAIIGLVKSDQICVGYHANNSTEQVDTIMEKNITVTHAQDILEKTHNGKLCNLNGVKPLILKDCSVAGWLLGNPMCDEFLNVSEWSYIVEKASPANGLCYPGDFNDYEELKHLLSRINHFE

NFDSNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNAPQIEGRRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGITNKINSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYEKVRLQLRDNAKELGNGCFEFYHKCDNECMESVKNGTYDYPQYSEEARLNREEISGVKLESIVTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICIckEgy_07: Already possesses the deletion in the 130 loop (SEQ ID NO: 73)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTQISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKRRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICIckEgy_07_LS: LS mutation on ckEgy_07 (SEQ ID NO: 74)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWG

ESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKRRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICIckEgy_07_H2RBS: 8 mutations (SEQ ID NO: 75)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHTASGVSRACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDEAEQRALYQNPTTQISVGTSTLNQRLVPKIATRSKVNGLGSRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKRRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI

Discussion

Several experimental studies have attempted to identify determinants ofhuman receptor specificity of H5 HA. However, these studies focused onreplacement of amino acids at the RBS sites directly without consideringthe influence of the neighboring positions. Further, no prior study haseven considered insertions or deletions as possible determinants of hostspecificity. Structure-based investigations have also fallen short ofidentifying the key determinants of H5 HA mainly because the structuraleffects of a deletion cannot be accurately evaluated using in silicoanalysis. In contrast, the present inventors, for the first time, haverecognized the importance of and have employed a sequence alignmentapproach to engineering proteins with novel properties. The presentinvention encompasses the recognition that a similar approach can beused for identifying determinants of host specificity of other HAsubtypes that have shown potential to infect humans in recent years (H7,H9, etc.).

Materials and Methods Dose Response Direct Binding of Wild Type HAPolypeptides to Glycans of Different Topology

Direct binding assays typically utilize glycan arrays in which definedglycan structures (e.g., monovalent or multivalent) are presented on asupport (e.g., glass slides or well plates), often using a polymerbackbone. In so-called “sequential” assays, trimeric HA polypeptide isbound to the array and then is detected, for example using labeled(e.g., with FITC or horse radish peroxidase) primary and secondaryantibodies. In “multivalent” assays, trimeric HA is first complexed withprimary and secondary antibodies (typically in a 4:2:1HA:primary:secondary ratio), such that there are 12 glycan binding sitesper pre-complexed HA, and is then contacted with the array. Bindingassays are typically carried out over a range of HA concentrations, sothat information is obtained regarding relative affinities for differentglycans in the array.

For example, direct binding studies were performed with arrays havingdifferent glycans such as 3′SLN, 6′SLN, 3′SLN-LN, 6′SLN-LN, and3′SLN-LN-LN, where LN represents Galβ1-4GlcNAc, 3′ representsNeu5Acα2-3, and 6′ represents Neu5Acα2-6). Specifically, biotinylatedglycans (50 μl of 120 pmol/ml) were incubated overnight (in PBS at 4°C.) with a streptavidin-coated High Binding Capacity 384-well plate thatwas previously rinsed three times with PBS. The plate was then washedthree times with PBS to remove excess glycan, and was used withoutfurther processing.

Appropriate amounts of His-tagged HA protein, primary antibody (mouseanti 6× His tag (SEQ ID NO: 207)) and secondary antibody (HRP conjugatedgoat anti-mouse IgG) were incubated in a ratio of 4:2:1HA:primary:secondary for 15 minutes on ice. The mixture (i.e.,precomplexed HA) was then made up to a final volume of 250 μl with 1%BSA in PBS. 50 μl of the precomplexed HA was then added to theglycan-coated wells in the 384-well plate, and was incubated at roomtemperature for 2 hours. The wells were subsequently washed three timeswith PBS containing 0.05% TWEEN-20, and then three times with PBS. HRPactivity was estimated using Amplex Red Peroxidase Kit (Invitrogen,Calif.) according to the manufacturer's instructions. Serial dilutionsof HA precomplexes were studied. Appropriate negative (non-sialylatedglycans) and background (no glycans or no HA) controls were included,and all assays were done in triplicate.

Example 2 Exemplary Human Binding H5 HA Polypeptide Variants

In some embodiments, HA polypeptides are H5 polypeptides. In some suchembodiments, H5 polypeptides in accordance with the invention showbinding (e.g., high affinity and/or specificity binding) to umbrellaglycans. In some such embodiments, H5 polypeptides in accordance withthe invention show either comparable (to umbrella topology binding) highaffinity-binding to cone topology glycans or reduced binding (e.g.,lower affinity and/or specificity relative to umbrella-topology glycans)to cone topology glycans.

In some embodiments, H5 HA polypeptides in accordance with the inventionbind to receptors found on human upper respiratory epithelial cells.Furthermore, H5 HA polypeptides in accordance with the invention bind toa plurality of different α2-6 sialylated glycans. In some embodiments,H5 HA polypeptides bind to umbrella glycans.

In some embodiments, H5 HA polypeptides in accordance with the inventionbind to HA receptors in the bronchus and/or trachea. In someembodiments, H5 HA polypeptides are not able to bind receptors in thedeep lung, and in some embodiments, H5 HA polypeptides are able to bindreceptors in the deep lung. In some embodiments, H5 HA polypeptides arenot able to bind to α2-3 sialylated glycans, and in some embodiments H5HA polypeptides are able to bind to α2-3 sialylated glycans.

In some embodiments, H5 HA polypeptides in accordance with the inventionare variants of a parent H5 HA (e.g., an H5 HA found in a naturalinfluenza isolate). For example, in some embodiments, H5 HA polypeptidesin accordance with the invention have at least one amino acidsubstitution, as compared with wild type H5 HA, within or affecting theglycan binding site. In some embodiments, such substitutions are ofamino acids that interact directly with bound glycan; in someembodiments, such substitutions are of amino acids that are one degreeof separation removed from those that interact with bound glycan, inthat the one degree of separation removed—amino acids either (1)interact with the direct-binding amino acids; (2) otherwise affect theability of the direct-binding amino acids to interact with glycan, butdo not interact directly with glycan themselves; or (3) otherwise affectthe ability of the direct-binding amino acids to interact with glycan,and also interact directly with glycan themselves. H5 HA polypeptides inaccordance with the invention contain substitutions of one or moredirect-binding amino acids, one or more first degree of separation—aminoacids, one or more second degree of separation—amino acids, or anycombination of these. In some embodiments, H5 HA polypeptides inaccordance with the invention may contain substitutions of one or moreamino acids with even higher degrees of separation.

In some embodiments, H5 HA polypeptide variants in accordance with theinvention have at least two, three, four, five or more amino acidsubstitutions as compared with wild type H5 HA; in some embodiments H5HA polypeptide variants in accordance with the invention have two,three, or four amino acid substitutions. In some embodiments, all suchamino acid substitutions are located within the glycan binding site.

In some embodiments, HA polypeptide variants in accordance with theinvention contain one or more amino acid substitutions as described inany of U.S. Patent Publication Number 2009/0269342 and 2010/0004195, andin U.S. patent application Ser. No. 12/829931, filed Jul. 2, 2010,entitled “COMPOSITIONS AND METHODS FOR DIAGNOSING AND/OR TREATINGINFLUENZA INFECTION” (all of which are incorporated herein byreference).

In some embodiments, H5 HA polypeptide variants have sequencesubstitutions at positions corresponding to one or more of residues 95,98, 128, 130, 131, 132, 133, 135, 136, 137, 138, 145, 153, 155, 156,158, 159, 160, 183, 186, 187, 188, 189, 190, 192, 193, 194, 195, 196,219, 221, 222, 224, 225, 226, 227, and 228. In some embodiments, H5 HApolypeptide variants have one or more amino acid substitutions relativeto a wild type parent H5 HA at residues selected from the groupconsisting of residues 95, 98, 128, 130, 131, 132, 133, 135, 136, 137,138, 145, 153, 155, 156, 158, 159, 160, 183, 186, 187, 188, 189, 190,192, 193, 194, 195, 196, 219, 221, 222, 224, 225, 226, 227, and 228. Insome embodiments, H5 HA polypeptide variants have one or more amino acidsubstitutions relative to a wild type parent H5 HA at any 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 residues selectedfrom the group consisting of residues 95, 98, 128, 130, 131, 132, 133,135, 136, 137, 138, 145, 153, 155, 156, 158, 159, 160, 183, 186, 187,188, 189, 190, 192, 193, 194, 195, 196, 219, 221, 222, 224, 225, 226,227, and 228.

In some embodiments, H5 HA polypeptide variants have sequencesubstitutions that reduce or abolish glycosylation a site correspondingto position 158. In some embodiments, H5 HA polypeptide variants havesequence substitutions that affect and/or alter the identity and/orstructure of the glycan linked to a site corresponding to position158.In some embodiments, such a sequence substitution is a mutation at asite corresponding to position 158, e.g., Asn158Xaa, wherein Xaa is anyamino acid other than Asn. In some embodiments, such a sequencesubstitution is a mutation at a site corresponding to position 160,e.g., Thr160Xaa, wherein Xaa is any amino acid other than Asn. In someembodiments, such a sequence substitution comprises the mutationThr160Ala. In some embodiments, a sequence substitution that reduces,abolishes, affects, or alters glycosylation at a site corresponding toposition 158 can make an H5 HA polypeptide more closely resemble (e.g.,both structurally and functionally) an H2 HA polypeptide. In someembodiments, a mutation at a site corresponding to position 160 (e.g.,Thr160Xaa, such as Thr160Ala) can make an H5 HA polypeptide more closelyresemble (e.g., both structurally and functionally) an H2 HApolypeptide.

In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent H5 HA at positionscorresponding to one or more of residues 226, 228, and 160. In someembodiments, an H5 HA polypeptide variant has one or more sequencesubstitutions relative to a wild type parent H5 HA at positionscorresponding to residues 226, 228, and 160. In some embodiments, an H5HA polypeptide variant has one or more sequence substitutions relativeto a wild type parent H5 HA at positions corresponding to residues 226and 160. In some embodiments, an H5 HA polypeptide variant has one ormore sequence substitutions relative to a wild type parent H5 HA atpositions corresponding to residues 228 and 160. In some embodiments, anH5 HA polypeptide variant has one or more sequence substitutionsrelative to a wild type parent H5 HA at positions corresponding toresidues 226 and 228.

In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent H5 HA at positionscorresponding to one or more of residues 226, 228, and 158. In someembodiments, an H5 HA polypeptide variant has one or more sequencesubstitutions relative to a wild type parent H5 HA at positionscorresponding to residues 226, 228, and 158. In some embodiments, an H5HA polypeptide variant has one or more sequence substitutions relativeto a wild type parent H5 HA at positions corresponding to residues 226and 158. In some embodiments, an H5 HA polypeptide variant has one ormore sequence substitutions relative to a wild type parent H5 HA atpositions corresponding to residues 228 and 158. In some embodiments, anH5 HA polypeptide variant has one or more sequence substitutionsrelative to a wild type parent H5 HA at positions corresponding toresidues 226 and 228.

In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions that include a deletion in one or more of theloop regions of an HA polypeptide. In some embodiments, an H5 HApolypeptide variant has one or more sequence substitutions that includea deletion at a site corresponding to the 128-137 loop region of an HApolypeptide. In some embodiments, an H5 HA polypeptide variant has oneor more sequence substitutions that include a deletion at one or more ofamino acid positions corresponding to residues 128, 129, 130, 131, 132,133, 134, 135, 136, and/or 137 of an HA polypeptide. In someembodiments, an H5 HA polypeptide variant has one or more sequencesubstitutions that include a deletion at a site corresponding to the128-134 loop region of an HA polypeptide. In some embodiments, an H5 HApolypeptide variant has one or more sequence substitutions that includea deletion at one or more of amino acid positions corresponding toresidues 128, 129, 130, 131, 132, 133, and/or 134 of an HA polypeptide.In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions that include a deletion of an amino acidcorresponding to residue 130. In some embodiments, such loop regionsubstitutions can make an H5 HA polypeptide more closely resemble (e.g.,both structurally and functionally) an H2 HA polypeptide. In someembodiments, a deletion of an amino acid corresponding to residue 130can make an H5 HA polypeptide more closely resemble (e.g., bothstructurally and functionally) an H2 HA polypeptide.

In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent HA at positionscorresponding to one or more of residues 131, 132, 133, 135, 137, 155,188, 192, 193, 221, 226, 227, 228, and 130. In some embodiments, an H5HA polypeptide variant has one or more sequence substitutions relativeto a wild type parent HA at positions corresponding to any 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of residues 131, 132, 133, 135,137, 155, 188, 192, 193, 221, 226, 227, 228, and 130. In someembodiments, an H5 HA polypeptide variant has one or more sequencesubstitutions relative to a wild type parent HA at positionscorresponding to (1) 130, and (2) one or more of residues 131, 132, 133,135, 137, 155, 188, 192, 193, 221, 226, 227, and 228. In someembodiments, an H5 HA polypeptide variant has one or more sequencesubstitutions relative to a wild type parent HA at positionscorresponding to (1) 130, and (2) any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, or 13 of residues 131, 132, 133, 135, 137, 155, 188, 192, 193, 221,226, 227, and 228.

In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent HA at positionscorresponding to one or more of residues 131, 132, 135, 188, 192, 221,and 130. In some embodiments, an H5 HA polypeptide variant has one ormore sequence substitutions relative to a wild type parent HA atpositions corresponding to any 1, 2, 3, 4, 5, 6, or 7 of residues 131,132, 135, 188, 192, 221, and 130. In some embodiments, an H5 HApolypeptide variant has one or more sequence substitutions relative to awild type parent HA at positions corresponding to (1) 130, and (2) oneor more of residues 131, 132, 135, 188, 192, and 221. In someembodiments, an H5 HA polypeptide variant has one or more sequencesubstitutions relative to a wild type parent HA at positionscorresponding to (1) 130, and (2) any 1, 2, 3, 4, 5, or 6 of residues131, 132, 135, 188, 192, and 221.

In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent HA at positionscorresponding to one or more of residues 133, 137, 155, 193, 226, 227,228, and 130. In some embodiments, an H5 HA polypeptide variant has oneor more sequence substitutions relative to a wild type parent HA atpositions corresponding to any 1, 2, 3, 4, 5, 6, 7, or of residues 133,137, 155, 193, 226, 227, 228, and 130. In some embodiments, an H5 HApolypeptide variant has one or more sequence substitutions relative to awild type parent HA at positions corresponding to (1) 130, and (2) oneor more of residues 133, 137, 155, 193, 226, 227, and 228. In someembodiments, an H5 HA polypeptide variant has one or more sequencesubstitutions relative to a wild type parent HA at positionscorresponding to (1) 130, and (2) any 1, 2, 3, 4, 5, 6, or 7 of residues133, 137, 155, 193, 226, 227, and 228.

In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent HA at positionscorresponding to one or more of residues 130, 192, and 193. In someembodiments, an H5 HA polypeptide variant has one or more sequencesubstitutions relative to a wild type parent HA at positionscorresponding to any 1, 2, or 3 of residues 130, 192, 193. In someembodiments, an H5 HA polypeptide variant has one or more sequencesubstitutions relative to a wild type parent HA at positionscorresponding to (1) 130, and (2) one or both of residues 192 and 193.

In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent HA at positionscorresponding to one or more of residues 131, 132, 133, 135, 137, 155,158, 160, 188, 192, 193, 221, 226, 227, 228, and 130. In someembodiments, an H5 HA polypeptide variant has one or more sequencesubstitutions relative to a wild type parent HA at positionscorresponding to any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,or 16 of residues 131, 132, 133, 135, 137, 155, 158, 160, 188, 192, 193,221, 226, 227, 228, and 130. In some embodiments, an H5 HA polypeptidevariant has one or more sequence substitutions relative to a wild typeparent HA at positions corresponding to (1) 130, and (2) one or more ofresidues 131, 132, 133, 135, 137, 155, 158, 160, 188, 192, 193, 221,226, 227, and 228. In some embodiments, an H5 HA polypeptide variant hasone or more sequence substitutions relative to a wild type parent HA atpositions corresponding to (1) 130, and (2) any 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, or 15 of residues 131, 132, 133, 135, 137, 155,158, 160, 188, 192, 193, 221, 226, 227, and 228.

In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent HA at positionscorresponding to one or more of residues 137, 188, 192, 193, 226, 228,and 130. In some embodiments, an H5 HA polypeptide variant has one ormore sequence substitutions relative to a wild type parent HA atpositions corresponding to any 1, 2, 3, 4, 5, 6, or 7 of residues 137,188, 192, 193, 226, 228, and 130. In some embodiments, an H5 HApolypeptide variant has one or more sequence substitutions relative to awild type parent HA at positions corresponding to (1) 130, and (2) oneor more of residues 137, 188, 192, 193, 226, and 228. In someembodiments, an H5 HA polypeptide variant has one or more sequencesubstitutions relative to a wild type parent HA at positionscorresponding to (1) 130, and (2) any 1, 2, 3, 4, 5, or 6 of residues137, 188, 192, 193, 226, and 228.

In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent HA at positionscorresponding to one or more of residues 137, 188, 192, 193, 226, 227,228, 131, 132, 133, and 130. In some embodiments, an H5 HA polypeptidevariant has one or more sequence substitutions relative to a wild typeparent HA at positions corresponding to any 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or 11 of residues 137, 188, 192, 193, 226, 227, 228, 131, 132, 133,and 130. In some embodiments, an H5 HA polypeptide variant has one ormore sequence substitutions relative to a wild type parent HA atpositions corresponding to (1) 130, and (2) one or more of residues 137,188, 192, 193, 226, 227, 228, 131, 132, and 133. In some embodiments, anH5 HA polypeptide variant has one or more sequence substitutionsrelative to a wild type parent HA at positions corresponding to (1) 130,and (2) any 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of residues 137, 188, 192,193, 226, 227, 228, 131, 132, and 133.

In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent HA at positionscorresponding to one or more of residues 227, 131, 132, 133, and 130. Insome embodiments, an H5 HA polypeptide variant has one or more sequencesubstitutions relative to a wild type parent HA at positionscorresponding to any 1, 2, 3, 4, or 5 of residues 227, 131, 132, 133,and 130. In some embodiments, an H5 HA polypeptide variant has one ormore sequence substitutions relative to a wild type parent HA atpositions corresponding to (1) 130, and (2) one or more of residues 227,131, 132, and 133. In some embodiments, an H5 HA polypeptide variant hasone or more sequence substitutions relative to a wild type parent HA atpositions corresponding to (1) 130, and (2) any 1, 2, 3, or 4 ofresidues 227, 131, 132, and 133.

In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent HA at positionscorresponding to one or more of residues 131, 133, 137, 155, 188, 192,193, 226, 227, 228, and 130. In some embodiments, an H5 HA polypeptidevariant has one or more sequence substitutions relative to a wild typeparent HA at positions corresponding to any 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or 11 of residues 131, 133, 137, 155, 188, 192, 193, 226, 227, 228,and 130. In some embodiments, an H5 HA polypeptide variant has one ormore sequence substitutions relative to a wild type parent HA atpositions corresponding to (1) 130, and (2) one or more of residues 131,133, 137, 155, 188, 192, 193, 226, 227, and 228. In some embodiments, anH5 HA polypeptide variant has one or more sequence substitutionsrelative to a wild type parent HA at positions corresponding to (1) 130,and (2) any 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of residues 131, 133, 137,155, 188, 192, 193, 226, 227, and 228.

In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent HA at positionscorresponding to one or more of residues 131, 133, 137, 155, 188, 192,193, 226, 228, and 130. In some embodiments, an H5 HA polypeptidevariant has one or more sequence substitutions relative to a wild typeparent HA at positions corresponding to any 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 of residues 131, 133, 137, 155, 188, 192, 193, 226, 228, and 130.In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent HA at positionscorresponding to (1) 130, and (2) one or more of residues 131, 133, 137,155, 188, 192, 193, 226, and 228. In some embodiments, an H5 HApolypeptide variant has one or more sequence substitutions relative to awild type parent HA at positions corresponding to (1) 130, and (2) any1, 2, 3, 4, 5, 6, 7, 8, or 9 of residues 131, 133, 137, 155, 188, 192,193, 226, and 228.

In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent HA at positionscorresponding to one or more of residues 131, 133, 137, 155, 159, 160,188, 192, 193, 226, 227, 228, and 130. In some embodiments, an H5 HApolypeptide variant has one or more sequence substitutions relative to awild type parent HA at positions corresponding to any 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, or 13 of residues 131, 133, 137, 155, 159, 160,188, 192, 193, 226, 227, 228, and 130. In some embodiments, an H5 HApolypeptide variant has one or more sequence substitutions relative to awild type parent HA at positions corresponding to (1) 130, and (2) oneor more of residues 131, 133, 137, 155, 159, 160, 188, 192, 193, 226,227, and 228. In some embodiments, an H5 HA polypeptide variant has oneor more sequence substitutions relative to a wild type parent HA atpositions corresponding to (1) 130, and (2) any 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 of residues 131, 133, 137, 155, 159, 160, 188, 192,193, 226, 227, and 228.

In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent HA at positionscorresponding to one or more of residues 131, 133, 137, 155, 159, 160,188, 192, 193, 226, 228, and 130. In some embodiments, an H5 HApolypeptide variant has one or more sequence substitutions relative to awild type parent HA at positions corresponding to any 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 of residues 131, 133, 137, 155, 159, 160, 188,192, 193, 226, 228, and 130. In some embodiments, an H5 HA polypeptidevariant has one or more sequence substitutions relative to a wild typeparent HA at positions corresponding to (1) 130, and (2) one or more ofresidues 131, 133, 137, 155, 159, 160, 188, 192, 193, 226, and 228. Insome embodiments, an H5 HA polypeptide variant has one or more sequencesubstitutions relative to a wild type parent HA at positionscorresponding to (1) 130, and (2) any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or11 of residues 131, 133, 137, 155, 159, 160, 188, 192, 193, 226, and228.

In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent HA at positionscorresponding to one or more of residues 137, 188, 192, 193, 226, 228,131, 132, 133, 221, 227, and 130. In some embodiments, an H5 HApolypeptide variant has one or more sequence substitutions relative to awild type parent HA at positions corresponding to any 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 of residues 137, 188, 192, 193, 226, 228, 131,132, 133, 221, 227, and 130. In some embodiments, an H5 HA polypeptidevariant has one or more sequence substitutions relative to a wild typeparent HA at positions corresponding to (1) 130, and (2) one or more ofresidues 137, 188, 192, 193, 226, 228, 131, 132, 133, 221, and 227. Insome embodiments, an H5 HA polypeptide variant has one or more sequencesubstitutions relative to a wild type parent HA at positionscorresponding to (1) 130, and (2) any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or11 of residues 137, 188, 192, 193, 226, 228, 131, 132, 133, 221, and227.

In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent HA at positionscorresponding to one or more of residues 131, 132, 133, 221, 227, and130. In some embodiments, an H5 HA polypeptide variant has one or moresequence substitutions relative to a wild type parent HA at positionscorresponding to any 1, 2, 3, 4, 5, or 6 of residues 131, 132, 133, 221,227, and 130. In some embodiments, an H5 HA polypeptide variant has oneor more sequence substitutions relative to a wild type parent HA atpositions corresponding to (1) 130, and (2) one or more of residues 131,132, 133, 221, and 227. In some embodiments, HA polypeptide variants(e.g., H5 HA polypeptide variants) have sequence substitutions relativeto a wild type parent HA at positions corresponding to (1) 130, and (2)any 1, 2, 3, 4, or 5 of residues 131, 132, 133, 221, and 227.

In some embodiments, a H5 HA polypeptide variant has one or more aminoacid substitutions relative to a wild type parent H5 HA at residuesselected from amino acids located in the region of the receptor thatdirectly binds to the glycan, including but not limited to residues 98,136, 153, 155, 183, and 194. In some embodiments, an H5 HA polypeptidevariant has one or more amino acid substitutions relative to a wild typeparent H5 HA at residues selected from amino acids located adjacent tothe region of the receptor that directly binds the glycan, including butnot limited to (a) residues 98 and 195, (b) residues 98, 138, 186, 187,195, and 228), or (c) residues 138, 186, 187, and 228.

In some embodiments, an HA polypeptide variant, and particularly an H5polypeptide variant has one or more amino acid substitutions relative toa wild type parent HA at residues selected from amino acids that are onedegree of separation removed from those that interact with bound glycan,in that the one degree of separation removed-amino acids either (1)interact with the direct-binding amino acids; (2) otherwise affect theability of the direct-binding amino acids to interact with glycan, butdo not interact directly with glycan themselves; or (3) otherwise affectthe ability of the direct-binding amino acids to interact with glycan,and also interact directly with glycan themselves, including but notlimited to residues 98, 138, 186, 187, 195, and 228.

In some embodiments, an HA polypeptide variant, and particularly an H5polypeptide variant, has one or more amino acid substitutions relativeto a wild type parent HA at residues selected from amino acids that areone degree of separation removed from those that interact with boundglycan, in that the one degree of separation removed-amino acids either(1) interact with the direct-binding amino acids; (2) otherwise affectthe ability of the direct-binding amino acids to interact with glycan,but do not interact directly with glycan themselves; or (3) otherwiseaffect the ability of the direct-binding amino acids to interact withglycan, and also interact directly with glycan themselves, including butnot limited to residues 138, 186, 187, and 228.

In some embodiments, an HA polypeptide variant, and particularly an H5polypeptide variant, has one or more amino acid substitutions relativeto a wild type parent HA at residues selected from amino acids that areone degree of separation removed from those that interact with boundglycan, in that the one degree of separation removed-amino acids either(1) interact with the direct-binding amino acids; (2) otherwise affectthe ability of the direct-binding amino acids to interact with glycan,but do not interact directly with glycan themselves; or (3) otherwiseaffect the ability of the direct-binding amino acids to interact withglycan, and also interact directly with glycan themselves, including butnot limited to residues 98 and 195.

In some embodiments, an HA polypeptide variant, and particularly an H5polypeptide variant, has an amino acid substitution relative to a wildtype parent HA at residue 159.

In some embodiments, an HA polypeptide variant, and particularly an H5polypeptide variant, has one or more amino acid substitutions relativeto a wild type parent HA at residues selected from 190, 193, 225, and226. In some embodiments, an HA polypeptide variant, and particularly anH5 polypeptide variant, has one or more amino acid substitutionsrelative to a wild type parent HA at residues selected from 190, 193,226, and 228.

In some embodiments, a H5 HA polypeptide variant in accordance with theinvention has one or more of the following amino acid substitutions:Ser132Thr, Ala133Thr, Ser133Thr, Ser137Ala, Ser137Arg, Ile155Thr,Lys156Glu, Asn158Xaa (wherein Xaa=any amino acid besides Asn),Thr160Ala, Asn186Pro, Asp187Ser, Asp187Thr, Ala188Glu, Ala188Asp,Ala189Gln, Ala189Lys, Ala189Thr, Glu190Asp, Glu190Thr, Thr192Arg/Lys,Lys193Arg, Lys193Asn, Lys193His, Lys193Ser, Lys/Arg193Thr/Ala/Met/Val,Ser221Pro, Gly225Asp, Gln226Ile, Gln226Leu, Gln226Val, Ser227Ala,Gly228Ser.

In some embodiments, an H5 HA polypeptide variant in accordance with theinvention has an amino acid substitution at a position corresponding toresidue 192, which switches the charge at that position. In someembodiments, an H5 HA polypeptide variant in accordance with theinvention has an amino acid substitution at a position corresponding toresidue 193, which switches the charge at that position. For example, insome embodiments, an H5 HA polypeptide in accordance with the inventionhas a Thr or a hydrophobic residue (e.g., Val or Ile) at a positioncorresponding to residue 192, and an H5 HA polypeptide variant (e.g., ahuman-adapted variant) has a hydrophilic residue at a positioncorresponding to residue 192. In some embodiments, an H5 HA polypeptidevariant (e.g., a human-adapted variant) has a hydrophilic residue at aposition corresponding to residue 192. To give another example, in someembodiments, an H5 HA polypeptide in accordance with the invention has aThr or a hydrophobic residue (e.g., Val or Ile) at a positioncorresponding to residue 192, and an H5 HA polypeptide variant (e.g., ahuman-adapted variant) has a basic residue (e.g., Lys or Arg) at aposition corresponding to residue 192. In some embodiments, an H5 HApolypeptide variant (e.g., a human-adapted variant) has a basic residue(e.g., Lys or Arg) at a position corresponding to residue 192. To giveyet another example, in some embodiments, an H5 HA polypeptide inaccordance with the invention has a basic residue (e.g., Lys or Arg) ata position corresponding to residue 193, and an H5 HA polypeptidevariant (e.g., a human-adapted variant) has a neutral or acidic residueat a position corresponding to residue 193. In some embodiments, an H5HA polypeptide variant (e.g., a human-adapted variant) has a neutral oracidic residue at a position corresponding to residue 193. In someembodiments, an H5 HA polypeptide variant (e.g., a human-adaptedvariant) has a Thr, Ala, Met, or Val at a position corresponding toresidue 193.

In some embodiments, human adaptation of an H5 HA polypeptide isassociated with the propert(ies) of the residue at position 188. In H5HA, residue 188 is frequently Ala, which makes contacts with Thr or ahydrophobic residue at 192. In contrast, in H2 HA, residue 188 isfrequently Glu or Asp, which makes contacts with Arg or Lys at 192.Hence, in some embodiments, an H5 HA polypeptide variant has a Glu atposition 188. In some embodiments, an H5 HA polypeptide variant has anAsp at position 188. In some embodiments, an H5 HA polypeptide varianthas an Ala188Glu substitution. In some embodiments, an H5 HA polypeptidevariant has an Ala188Asp substitution.

In some embodiments, an H5 HA polypeptide has an Ala at a positioncorresponding to residue 188 and a Thr at a position corresponding toresidue 192. In some embodiments, an H5 HA polypeptide has an Ala at aposition corresponding to residue 188 and a hydrophobic residue at aposition corresponding to residue 192. In some embodiments, an H5 HApolypeptide variant has a Glu at a position corresponding to residue 188and an Arg at a position corresponding to residue 192. In someembodiments, an H5 HA polypeptide variant has an Asp at a positioncorresponding to residue 188 and an Arg at a position corresponding toresidue 192. In some embodiments, an H5 HA polypeptide variant has a Gluat a position corresponding to residue 188 and a Lys at a positioncorresponding to residue 192. In some embodiments, an H5 HA polypeptidevariant has an Asp at a position corresponding to residue 188 and a Lysat a position corresponding to residue 192.

In some embodiments, an H5 HA polypeptide has an Ala at a positioncorresponding to residue 188, a Thr at a position corresponding toresidue 192, and a Lys at a position corresponding to residue 193. Insome embodiments, an H5 HA polypeptide has an Ala at a positioncorresponding to residue 188, a hydrophobic residue at a positioncorresponding to residue 192, and a Lys at a position corresponding toresidue 193. In some embodiments, an H5 HA polypeptide has an Ala at aposition corresponding to residue 188, a Thr at a position correspondingto residue 192, and an Arg at a position corresponding to residue 193.In some embodiments, an H5 HA polypeptide has an Ala at a positioncorresponding to residue 188, a hydrophobic residue at a positioncorresponding to residue 192, and an Arg at a position corresponding toresidue 193. In some embodiments, an H5 HA polypeptide variant has a Gluat a position corresponding to residue 188, an Arg at a positioncorresponding to residue 192, and a Thr at a position corresponding toresidue 193. In some embodiments, an H5 HA polypeptide variant has anAsp at a position corresponding to residue 188, an Arg at a positioncorresponding to residue 192, and a Thr at a position corresponding toresidue 193. In some embodiments, an H5 HA polypeptide variant has a Gluat a position corresponding to residue 188, a Lys at a positioncorresponding to residue 192, and a Thr at a position corresponding toresidue 193. In some embodiments, an H5 HA polypeptide variant has anAsp at a position corresponding to residue 188, a Lys at a positioncorresponding to residue 192, and a Thr at a position corresponding toresidue 193. In some embodiments, an H5 HA polypeptide variant has a Gluat a position corresponding to residue 188, an Arg at a positioncorresponding to residue 192, and a Thr, Ala, Met, or Val at a positioncorresponding to residue 193. In some embodiments, an H5 HA polypeptidevariant has an Asp at a position corresponding to residue 188, an Arg ata position corresponding to residue 192, and a Thr, Ala, Met, or Val ata position corresponding to residue 193. In some embodiments, an H5 HApolypeptide variant has a Glu at a position corresponding to residue188, a Lys at a position corresponding to residue 192, and a Thr, Ala,Met, or Val at a position corresponding to residue 193. In someembodiments, an H5 HA polypeptide variant has an Asp at a positioncorresponding to residue 188, a Lys at a position corresponding toresidue 192, and a Thr, Ala, Met, or Val at a position corresponding toresidue 193.

In some embodiments, an H5 HA polypeptide has an Ala at a positioncorresponding to residue 131. In some embodiments, an H5 HA polypeptidevariant has a Thr at a position corresponding to residue 131.

In some embodiments, an H5 HA polypeptide has a Ser at a positioncorresponding to residue 132. In some embodiments, an H5 HA polypeptidevariant has a Thr at a position corresponding to residue 132.

In some embodiments, an H5 HA polypeptide has a Ser at a positioncorresponding to residue 133. In some embodiments, an H5 HA polypeptidevariant has a Thr at a position corresponding to residue 133.

In some embodiments, an H5 HA polypeptide includes Ala, Thr, and/or Serat any position corresponding to residues 131, 132, and/or 133. In someembodiments, an H5 HA polypeptide variant includes Ala, Thr, and/or Serat any position corresponding to residues 131, 132, and/or 133. In someembodiments, an H5 HA polypeptide variant includes Thr at all ofpositions corresponding to residues 131, 132, and 133.

In some embodiments, an H5 HA polypeptide has a Val at a positioncorresponding to residue 135. In some embodiments, an H5 HA polypeptidevariant has any amino acid other than Val at a position corresponding toresidue 135.

In some embodiments, an H5 HA polypeptide has a Ser at a positioncorresponding to residue 137. In some embodiments, an H5 HA polypeptidevariant has an Arg at a position corresponding to residue 137.

In some embodiments, an H5 HA polypeptide has an Ile at a positioncorresponding to residue 155. In some embodiments, an H5 HA polypeptidevariant has a Thr at a position corresponding to residue 155. In someembodiments, an H5 HA polypeptide includes a Thr at a positioncorresponding to residue 155. In some embodiments, an H5 HA polypeptidevariant includes a Thr at a position corresponding to residue 155.

In some embodiments, an H5 HA polypeptide has a Ser at a positioncorresponding to residue 221. In some embodiments, an H5 HA polypeptidevariant has a Pro at a position corresponding to residue 221.

In some embodiments, an H5 HA polypeptide includes a Ser at a positioncorresponding to residue 221. In some embodiments, an H5 HA polypeptidevariant includes a Pro at a position corresponding to residue 221.Without wishing to be bound by any one particular theory, Pro221 mightinfluence conformation of 220 loop which is involved with the RBS of H2HA.

In some embodiments, an H5 HA polypeptide has a Gln at a positioncorresponding to residue 226. In some embodiments, an H5 HA polypeptidevariant has a Leu at a position corresponding to residue 226.

In some embodiments, an H5 HA polypeptide has a Ser at a positioncorresponding to residue 227. In some embodiments, an H5 HA polypeptidevariant has a Gly at a position corresponding to residue 227.

In some embodiments, an H5 HA polypeptide has a Gly at a positioncorresponding to residue 228. In some embodiments, an H5 HA polypeptidevariant has a Ser at a position corresponding to residue 228.

In some embodiments, an H5 HA polypeptide includes Gln, Ser, and Glyresidues at positions 226, 227, and 228, respectively. In someembodiments, an H5 HA polypeptide variant includes a Leu, Gly, and Serat positions 226, 227, and 228, respectively.

In some embodiments, a H5 HA polypeptide variant in accordance with theinvention has one or more of the following amino acids at the indicatedpositions:

-   -   Glu190Asp, Lys193Ser, Gly225Asp, Gln226Leu    -   Glu190Asp, Lys193Ser, Gln226Leu, Gly228Ser    -   Ala189Gln, Lys193Ser, Thr160Ala    -   Ala189Gln, Lys193Ser, Gln226Leu, Gly228Ser    -   Asp187Ser/Thr, Ala189Gln, Lys193Ser, Gln226Leu, Gly228Ser    -   Ala189Lys, Lys193Asn, Gln226Leu, Gly228Ser    -   Asp187Ser/Thr, Ala189Lys, Lys193Asn, Gln226Leu, Gly228Ser    -   Lys156Glu, Ala189Lys, Lys193Asn, Gln226Leu, Gly228Ser    -   Lys193His, Gln226Leu/Ile/Val, Gly228Ser    -   Lys193Arg, Gln226Leu/Ile/Val, Gly228Ser    -   Ala189Lys, Lys193Asn, Gly225Asp    -   Lys156Glu, Ala189Lys, Lys193Asn, Gly225Asp    -   Ser137Ala, Lys156Glu, Ala189Lys, Lys193Asn, Gly225Asp    -   Glu190Thr, Lys193Ser, Gly225Asp    -   Asp187Thr, Ala189Thr, Glu190Asp, Lys193Ser, Gly225Asp    -   Asn186Pro, Asp187Thr, Ala189Thr, Glu190Asp, Lys193Ser, Gly225Asp    -   Asn186Pro, Asp187Thr, Ala189Thr, Glu190Asp, Lys193Ser,        Gly225Asp, Ser227Ala    -   Gln226Leu, Gly228Ser, Thr160Ala    -   Gln226Leu, Gly228Ser, Thr160Ala    -   Gly228Ser, Thr160Ala    -   Gln226Leu, Thr160Ala    -   Gln226Leu, Gly228Ser    -   Thr160Ala    -   Gln226Leu, Gly228Ser, Asn158Xaa (wherein Xaa=any amino acid        besides Asn)    -   Gly228Ser, Asn158Xaa    -   Gln226Leu, Asn158Xaa    -   Gln226Leu, Gly228Ser    -   Asn158Xaa    -   Δ130 (wherein “Δ130” indicates a deletion at an amino acid        corresponding to position 130) plus any possible combination of        mutations at positions corresponding to 131, 132, 133, 135, 137,        155, 188, 192, 193, 221, 226, 227, and 228    -   Δ130 plus any possible combination of mutations at positions        corresponding to 131, 132, 135, 188, 192, and 221    -   Δ130 plus any possible combination of mutations at positions        corresponding to 133, 137, 155, 193, 226, 227, and 228    -   Δ130 plus any possible combination of mutations at positions        corresponding to 131, 132, 133, 135, 137, 155, 158, 160, 188,        192, 193, 221, 226, 227, and 228    -   Δ130 plus any possible combination of mutations at positions        corresponding to 131, 133, 137, 155, 188, 192, 193, 226, 227,        and 228    -   Δ130 plus any possible combination of mutations at positions        corresponding to 131, 133, 137, 155, 188, 192, 193, 226, and 228    -   Δ130 plus any possible combination of mutations at positions        corresponding to 131, 133, 137, 155, 159, 160, 188, 192, 193,        226, 227, and 228    -   Δ130 plus any possible combination of mutations at positions        corresponding to 131, 133, 137, 155, 159, 160, 188, 192, 193,        226, and 228    -   Δ130 plus any possible combination of mutations at positions        corresponding to 137, 188, 192, 193, 226, 228, 131, 132, 133,        221, and 227    -   Δ130 plus any possible combination of mutations at positions        corresponding to 131, 132, 133, 221, and 227    -   Δ130 plus any possible combination of mutations at positions        corresponding to 137, 188, 192, 193, 226, and 228    -   Δ130 plus any possible combination of mutations at positions        corresponding to 137, 188, 192, 193, 226, 227, 228, 131, 132,        and 133    -   Δ130 plus any possible combination of mutations at positions        corresponding to 227, 131, 132, and 133    -   Gln226Leu, Gly228Ser, Thr160Ala, Δ130    -   Gln226Leu, Gly228Ser, Δ130    -   Gln226Leu, Thr160Ala, Δ130    -   Gly228Ser, Thr160Ala, Δ130    -   Gln226Leu, Δ130    -   Gly228Ser, Δ130    -   Thr160Ala, Δ130    -   Δ130    -   Δ130, Ala131Thr, Leu133Thr, Ser137Arg, Ile155Thr, Ala188Glu,        Thr/Ile192Arg/Lys, Arg/Lys193Thr/Ala, Gln226Leu, Ser227Gly,        Gly228Ser    -   Δ130, Ala131Thr, Leu133Thr, Ser137Arg, Ile155Thr, Ala188Glu,        Thr/Ile192Arg/Lys, Arg/Lys193Thr/Ala, Gln226Leu, Gly228Ser    -   Δ130, Ala131Thr, Leu133Thr, Ser137Arg, Ile155Thr, Asn159Asp (or        Thr160Ala or both), Ala188Glu, Thr/Ile192Arg/Lys,        Arg/Lys193Thr/Ala, Gln226Leu, Ser227Gly, Gly228Ser    -   Δ130, Ala131Thr, Leu133Thr, Ser137Arg, Ile155Thr, Asn159Asp (or        Thr160Ala or both), Ala188Glu, Thr/Ile192Arg/Lys,        Arg/Lys193Thr/Ala, Gln226Leu, Gly228Ser    -   Δ130, Ser137Arg, Ala188Glu, Thr192Arg/Lys,        Arg/Lys193Thr/Met/Ala/Val, Gln226Leu, Gly228Ser    -   Δ130, Ser137Arg, Ala188Glu, Thr192Arg/Lys,        Arg/Lys193Thr/Met/Ala/Val, Gln226Leu, Gly228Ser, Xaa131Ser/Thr,        Xaa132Ser/Thr, Xaa133Ser/Thr, Ser221Pro, Ser227Gly (wherein        Xaa=any amino acid)    -   Δ130, Xaa131Ser/Thr, Xaa132Ser/Thr, Xaa133Ser/Thr, Ser221Pro,        Ser227Gly (wherein Xaa=any amino acid)    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid)    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue)    -   Δ130, Xaa193Xaa′ (wherein Xaa=a basic residue, e.g., Lys or Arg,        and Xaa′=a neutral or acidic residue)    -   Δ130, Lys/Arg193Thr/Ala/Met/Val    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid), Xaa193Xaa′ (wherein Xaa=a        basic residue, e.g., Lys or Arg, and Xaa′=a neutral or acidic        residue)    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue),        Xaa193Xaa′ (wherein Xaa=a basic residue, e.g., Lys or Arg, and        Xaa′=a neutral or acidic residue)    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid), Lys/Arg193Thr/Ala/Met/Val    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue),        Lys/Arg193Thr/Ala/Met/Val    -   Δ130, Ala188Glu    -   Δ130, Ala188Asp    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid), Ala188Glu    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue),        Ala188Glu    -   Δ130, Xaa193Xaa′ (wherein Xaa=a basic residue, e.g., Lys or Arg,        and Xaa′=a neutral or acidic residue), Ala188Glu    -   Δ130, Lys/Arg193Thr/Ala/Met/Val, Ala188Glu    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid), Ala188Asp    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue),        Ala188Asp    -   Δ130, Xaa193Xaa′ (wherein Xaa=a basic residue, e.g., Lys or Arg,        and Xaa′=a neutral or acidic residue), Ala188Asp    -   Δ130, Lys/Arg193Thr/Ala/Met/Val, Ala188Asp    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid), Xaa193Xaa′ (wherein Xaa=a        basic residue, e.g., Lys or Arg, and Xaa′=a neutral or acidic        residue), Ala188Glu    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue),        Xaa193Xaa′ (wherein Xaa=a basic residue, e.g., Lys or Arg, and        Xaa′=a neutral or acidic residue), Ala188Glu    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid), Lys/Arg193Thr/Ala/Met/Val,        Ala188Glu    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue),        Lys/Arg193Thr/Ala/Met/Val, Ala188Glu    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid), Xaa193Xaa′ (wherein Xaa=a        basic residue, e.g., Lys or Arg, and Xaa′=a neutral or acidic        residue), Ala188Asp    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue),        Xaa193Xaa′ (wherein Xaa=a basic residue, e.g., Lys or Arg, and        Xaa′=a neutral or acidic residue), Ala188Asp    -   Δ130, Xaa192Xaa′ (wherein Xaa=any hydrophobic amino acid, and        Xaa′=any hydrophilic amino acid), Lys/Arg193Thr/Ala/Met/Val,        Ala188Asp    -   Δ130, Xaa192Lys/Arg (wherein Xaa=any hydrophobic residue),        Lys/Arg193Thr/Ala/Met/Val, Ala188Asp

In some embodiments, the present invention provides H5 HA polypeptides(e.g., H5 HA polypeptide variants, engineered H5 HA polypeptides, and/orengineered H5 HA polypeptide variants) whose amino acid sequenceincludes an element as set forth below (the numbering of these positionscorresponds to the numbering of H3 HA):

-   -   X190, X193, X225 and X226    -   X190, X193, X226 and X228    -   X189, X193, X160    -   X189, X193, X226, X228    -   X187, X189, X193, X226, X228    -   X189, X193, X226, X228    -   X187, X189, X193, X226, X228    -   X156, X189, X193, X226, X228    -   X193, X226, X228    -   X193, X226, X228    -   X189, X193, X225    -   X156, X189, X193, X225    -   X137, X156, X189, X193, X225    -   X190, X193, X225    -   X187, X189, X190, X193, X225    -   X186, X187, X189, X190, X193, X225    -   X186, X187, X189, X190, X193, X225, X227    -   X226, X228, X160    -   X226, X228, X160    -   X228, X160    -   X226, X160    -   X226, X228    -   X160    -   X226, X228, Xaa158 (wherein Xaa=any amino acid besides Asn)    -   X228, Xaa158 (wherein Xaa=any amino acid besides Asn)    -   X226, Xaa158 (wherein Xaa=any amino acid besides Asn)    -   X226, X228    -   X158 (wherein Xaa=any amino acid besides Asn)    -   X130 plus any possible combination of X131, X132, X133, X135,        X137, X155, X188, X192, X193, X221, X226, X227, and X228    -   X130 plus any possible combination of X131, X132, X135, X188,        X192, and X221    -   X130 plus any possible combination of X133, X137, X155, X193,        X226, X227, and X228    -   X130 plus any possible combination of X131, X132, X133, X135,        X137, X155, Xaa158 (wherein Xaa=any amino acid besides Asn),        X160, X188, X192, X193, X221, X226, X227, and X228    -   X130 plus any possible combination of X131, X133, X137, X155,        X188, X192, X193, X226, X227, and X228    -   X130 plus any possible combination of X131, X133, X137, X155,        X188, X192, X193, X226, and X228    -   X130 plus any possible combination of X131, X133, X137, X155,        X159, X160, X188, X192, X193, X226, X227, and X228    -   X130 plus any possible combination of X131, X133, X137, X155,        X159, X160, X188, X192, X193, X226, and X228    -   X130 plus any possible combination of X137, X188, X192, X193,        X226, X228, X131, X132, X133, X221, and X227    -   X130 plus any possible combination of X131, X132, X133, X221,        and X227    -   X130 plus any possible combination of X137, X188, X192, X193,        X226, and X228    -   X130 plus any possible combination of X137, X188, X192, X193,        X226, X227, X228, X131, X132, and X133    -   X130 plus any possible combination of X227, X131, X132, and X133    -   X226, X228, X160, X130    -   X226, X228, X130    -   X226, X160, X130    -   X228, X160, X130    -   X226, X130    -   X228, X130    -   X160, X130    -   X130    -   X130, X131, X133, X137, X155, X188, X192, X193, X226, X227, X228    -   X130, X131, X133, X137, X155, X188, X192, X193, X226, X228    -   X130, X131, X133, X137, X155, X159, X160, X188, X192, X193,        X226, X227, X228    -   X130, X131, X133, X137, X155, X159, X160, X188, X192, X193,        X226, X228    -   X130, X137, X188, X192, X193, X226, X228    -   X130, X137, X188, X192, X193, X226, X228, X131, X132, X133,        X221, X227    -   X130, X131, X132, X133, X221, X227    -   X130, X192    -   X130, X193    -   X130, X192, X193    -   X130, X188    -   X130, X192, X188    -   X130, X193, X188    -   X130, X192, X193, X188

wherein X=any amino acid (unless otherwise specified above), and/or X=amissing amino acid. The numbering of these positions corresponds to thenumbering of H3 HA.

In some embodiments X130 is a deletion at at a site corresponding toposition 130. In some embodiments, X160 is an Ala. In some embodiments,X158 is any amino acid other than Asn.

In some such embodiments, the H5 HA polypeptide variant has at least onefurther substitution as compared with a wild type H5 HA, such thataffinity and/or specificity of the variant for umbrella glycans isincreased.

In some embodiments, H5 HA polypeptide variants in accordance with theinvention (including H5 HA polypeptide variants) have sequences thatinclude L226, S228, and A160. In some embodiments, H5 HA polypeptidevariants in accordance with the invention (including H5 HA polypeptidevariants) have sequences that include L226 and A160. In someembodiments, H5 HA polypeptide variants in accordance with the invention(including H5 HA polypeptide variants) have sequences that include S228and A160. In some embodiments, H5 HA polypeptide variants in accordancewith the invention (including H5 HA polypeptide variants) have sequencesthat include A160.

In some embodiments, H5 HA polypeptide variants in accordance with theinvention (including H5 HA polypeptide variants) have sequences thatinclude L226, S228, and X158 (wherein X=any amino acid besides Asn). Insome embodiments, H5 HA polypeptide variants in accordance with theinvention (including H5 HA polypeptide variants) have sequences thatinclude L226 and X158. In some embodiments, H5 HA polypeptide variantsin accordance with the invention (including H5 HA polypeptide variants)have sequences that include S228 and X158. In some embodiments, H5 HApolypeptide variants in accordance with the invention (including H5 HApolypeptide variants) have sequences that include X158.

In some embodiments, H5 HA polypeptide variants in accordance with theinvention (including H5 HA polypeptide variants) have sequences thatinclude Δ130 and any possible combination of mutations at positionscorresponding to 131, 132, 133, 135, 137, 155, 158, 160, 188, 192, 193,221, 226, 227, and 228.

In some embodiments, H5 HA polypeptide variants in accordance with theinvention (including H5 HA polypeptide variants) have sequences thatinclude Δ130, L226, S228, A160 and any possible combination of mutationsat positions corresponding to 131, 132, 133, 135, 137, 155, 158, 188,192, 193, 221, and 227. In some embodiments, H5 HA polypeptide variantsin accordance with the invention (including H5 HA polypeptide variants)have sequences that include Δ130, L226, A160, and any possiblecombination of mutations at positions corresponding to 131, 132, 133,135, 137, 155, 158, 188, 192, 193, 221, 227, and 228. In someembodiments, H5 HA polypeptide variants in accordance with the invention(including H5 HA polypeptide variants) have sequences that include Δ130,S228, A160, and any possible combination of mutations at positionscorresponding to 131, 132, 133, 135, 137, 155, 158, 188, 192, 193, 221,and 227. In some embodiments, H5 HA polypeptide variants in accordancewith the invention (including H5 HA polypeptide variants) have sequencesthat include Δ130, L226, S228, and any possible combination of mutationsat positions corresponding to 131, 132, 133, 135, 137, 155, 158, 160,188, 192, 193, 221, and 227.

In some embodiments, H5 HA polypeptide variants in accordance with theinvention (including H5 HA polypeptide variants) have sequences thatinclude Δ130, A160, and any possible combination of mutations atpositions corresponding to 131, 132, 133, 135, 137, 155, 158, 188, 192,193, 221, 226, 227, and 228. In some embodiments, H5 HA polypeptidevariants in accordance with the invention (including H5 HA polypeptidevariants) have sequences that include Δ130, L226, and any possiblecombination of mutations at positions corresponding to 131, 132, 133,135, 137, 155, 158, 160, 188, 192, 193, 221, and 227, and 228. In someembodiments, H5 HA polypeptide variants in accordance with the invention(including H5 HA polypeptide variants) have sequences that include Δ130,S228, and any possible combination of mutations at positionscorresponding to 131, 132, 133, 135, 137, 155, 158, 160, 188, 192, 193,221, 226, and 227.

In some embodiments, H5 HA polypeptides in accordance with the invention(including H5 HA polypeptide variants) have sequences that include Δ130,L226, S228, X158 (wherein X=any amino acid besides Asn) and any possiblecombination of mutations at positions corresponding to 131, 132, 133,135, 137, 155, 160, 188, 192, 193, 221, and 227. In some embodiments, H5HA polypeptides in accordance with the invention (including H5 HApolypeptide variants) have sequences that include Δ130, L226, X158, andany possible combination of mutations at positions corresponding to 131,132, 133, 135, 137, 155, 160, 188, 192, 193, 221, 227, and 228. In someembodiments, H5 HA polypeptides in accordance with the invention(including H5 HA polypeptide variants) have sequences that include Δ130,S228, X158, and any possible combination of mutations at positionscorresponding to 131, 132, 133, 135, 137, 155, 160, 188, 192, 193, 221,and 227. In some embodiments, H5 HA polypeptides in accordance with theinvention (including H5 HA polypeptide variants) have sequences thatinclude Δ130, L226, S228, and any possible combination of mutations atpositions corresponding to 131, 132, 133, 135, 137, 155, 158, 160, 188,192, 193, 221, and 227.

In some embodiments, H5 HA polypeptides in accordance with the invention(including H5 HA polypeptide variants) have sequences that include Δ130,X158 (wherein X=any amino acid besides Asn), and any possiblecombination of mutations at positions corresponding to 131, 132, 133,135, 137, 155, 160, 188, 192, 193, 221, 226, 227, and 228.

In some embodiments, H5 HA polypeptide variants in accordance with theinvention have an open binding site as compared with a parent H5 HA, andparticularly with a parent wild type H5 HAs.

In some embodiments, H5 HA polypeptides in accordance with the inventionbind to the following α2-6 sialylated glycans:

and combinations thereof. In some embodiments, H5 HA polypeptides inaccordance with the invention bind to glycans of the structure:

and combinations thereof; and/or

and combinations thereof. In some embodiments, H5 HA polypeptides inaccordance with the invention bind to

in some embodiments to in some embodiments

in some embodiments to

and in some embodiments to

In some embodiments, H5 HA polypeptides in accordance with the inventionbind to umbrella topology glycans. In some embodiments, H5 HApolypeptides in accordance with the invention bind to at least some ofthe glycans (e.g., α2-6 sialylated glycans) depicted in FIGS. 9A-9B. Insome embodiments, H5 HA polypeptides in accordance with the inventionbind to multiple glycans depicted in FIGS. 9A-9B.

In some embodiments, H5 HA polypeptides in accordance with the inventionbind to at least about 10%, about 15%, about 20%, about 25%, about 30%about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90% about 95%, ormore of the glycans found on HA receptors in human upper respiratorytract tissues (e.g., epithelial cells).

In some embodiments, H5 HA polypeptides (including H5 HA polypeptidevariants) are any of those set forth in SEQ ID NOs: 50, 51, 53-55,-60-75and 205-206.

In one aspect, the present invention provides the particular recognitionthat high affinity binding to umbrella-topology glycans alone may not besufficient to confer effective transmission to/infectivity of humans.The present invention provides the insight that reduced binding tocone-topology glycans may also be important. In some embodiments, highaffinity binding to umbrella-topology glycans and reduced affinitybinding to cone-topology glycans may be involved in conferring effectivetransmission to/infectivity of humans. In some embodiments, highaffinity binding to umbrella-topology glycans is sufficient to confereffective transmission to/infectivity of humans. In some embodiments,high affinity binding to umbrella-topology glycans is sufficient toconfer effective transmission to/infectivity of humans, even if theaffinity of binding to cone-topology glycans is not reduced (e.g.,unchanged, increased, etc.).

In some embodiments, increased affinity and/or specificity of binding ofan H5 HA polypeptide variant to umbrella-topology glycans and reducedaffinity and/or specificity binding to cone-topology glycans may beinvolved in increasing or enhancing transmission to/infectivity ofhumans relative to a reference polypeptide (e.g., the H5 HA polypeptidevariant's cognate parent HA polypeptide). In some embodiments, increasedaffinity and/or specificity of binding of an H5 HA polypeptide variantto umbrella-topology glycans is sufficient to increase or enhancetransmission to/infectivity of humans relative to a referencepolypeptide (e.g., the H5 HA polypeptide variant's cognate parent HApolypeptide). In some embodiments, increased affinity and/or specificityof binding of an H5 HA polypeptide variant to umbrella-topology glycansis sufficient to increase or enhance transmission to/infectivity ofhumans relative to a reference polypeptide (e.g., the H5 HA polypeptidevariant's cognate parent HA polypeptide), even if the affinity and/orspecificity of binding to cone-topology glycans is not reduced (e.g.,unchanged, increased, etc.). In some embodiments, increased affinityand/or specificity of binding of an H5 HA polypeptide variant toumbrella-topology glycans is sufficient to increase or enhancetransmission to/infectivity of humans relative to a referencepolypeptide (e.g., the H5 HA polypeptide variant's cognate parent HApolypeptide), even if the affinity and/or specificity of binding tocone-topology glycans is equal to and/or greater than that of theaffinity and/or specificity of binding to umbrella-topology glycans.

Example 3 Glycan Diversity in Human Upper Respiratory Tissues

Lectin binding studies showed diversity in the distribution of α2-3 andα2-6 in the upper respiratory tissues. Staining studies indicatepredominant distribution of α2-6 sialylated glycans as a part of bothN-linked (ciliated cells) and O-linked glycans (in the goblet cells) onthe apical side of the tracheal epithelium (FIG. 12). On the other hand,the internal regions of the tracheal tissue predominantly comprises ofα2-3 distributed on N-linked glycans.

MALDI-MS glycan profiling analyses showed a substantial diversity (FIG.10) as well as predominant expression of α2-6 sialylated glycans on thehuman upper airways. Fragmentation of representative mass peaks usingMALDI TOF-TOF supports glycan topologies where longer oligosaccharidebranches with multiple lactosamine repeats are extensively distributedas compared to short oligosaccharide branches (FIG. 10). To provide areference for the diversity in the distribution and topology of glycansin the upper airway, MALDI-MS analysis was performed on N-linked glycansfrom human colonic epithelial cells (HT29). Recent H5N1 viruses haveprimarily infected the gut, and hence, these cells were chosen asrepresentative gut cells. The glycan profile of HT29 cells issignificantly different from that of the HBEs wherein there is apredominant distribution of α2-3 and the long oligosaccharide branchglycan topology is not as observed (FIG. 10).

Data in FIG. 12 were generated by the following method. Formalin fixedand paraffin embedded human tracheal tissue sections were purchased fromUS Biological. After the tissue sections were deparaffinized andrehydrated, endogenous biotin was blocked using the streptavidin/biotinblocking kit (Vector Labs). Sections were then incubated with FITClabeled Jacalin (specific for O-linked glycans), biotinylatedConcanavalin A (Con A, specific for α-linked mannose residues, which arepart of the core oligosaccharide structure that constitute N-linkedglycans), biotinylated Maackia amurensis lectin (MAL, specific forSAα2,3-gal) and biotinylated Sambuccus nigra agglutinin (SNA, specificfor SAα2,6-gal) (Vector labs; 10 μg/ml in PBS with 0.5% Tween-20) for 3hrs. After washing with TBST (Tris buffered saline with 1% Tween-20),the sections were incubated with Alexa fluor 546 streptavidin (2 μg/mlin PBS with 0.5% Tween-20) for 1 hr. Slides were washed with TBST andviewed under a confocal microscope (Zeiss LSM510 laser scanning confocalmicroscopy). All incubations were performed at room temperature (RT).

Data in FIG. 10 were generated using the following method. The cells(˜70×10⁶) were harvested when they were >90% confluent with 100 mMcitrate saline buffer and the cell membrane was isolated after treatmentwith protease inhibitor (Calbiochem) and homogenization. The cellmembrane fraction was treated with PNGaseF (New England Biolabs) and thereaction mixture was incubated overnight at 37° C. The reaction mixturewas boiled for 10 min to deactivate the enzyme and the deglycosylatedpeptides and proteins were removed using a Sep-Pak C18 SPE cartridge(Waters). Glycans were further desalted and purified into neutral (25%acetonitrile fraction) and acidic (50% acetonitrile containing 0.05%trifluoroacetic acid) fractions using graphitized carbon solid—phaseextraction columns (Supelco). Acidic fractions were analyzed byMALDI-TOF MS in positive and negative modes respectively with softionization conditions (accelerating voltage 22 kV, grid voltage 93%,guide wire 0.3% and extraction delay time of 150 ns). The peaks werecalibrated as non-sodiated species. The predominant expression of α2-6sialylated glycans was confirmed by pretreatment of samples usingSialidase A and S. Isolated glycans were subsequently incubated with 0.1U of Arthrobacter ureafaciens sialidase (Sialidase A, non-specific) orStreptococcus pneumoniae sialidase (Sialidase S, specific for α2-3sialylated glycans) in a final volume of 100 mL of 50 mM sodiumphosphate, pH 6.0 at 37° C. for 24 hrs. Neutral and the acidic fractionswere analyzed by MALDI-TOF MS in positive and negative modesrespectively.

Example 4 Binding of H5 HA Polypeptide Variants to Human Lung Tissues

Binding of Formalin fixed and paraffin embedded human tissue lung andtracheal sections are provided (e.g., purchased from US Biomax, Inc. andfrom US Biological, respectively). Tissue sections are deparaffinized,rehydrated and incubated with 1% BSA in PBS for 30 minutes to preventnon-specific binding. H5 HAs are pre-complexed with primary antibody(mouse anti 6× His tag (SEQ ID NO: 207)) and secondary antibody(Alexa-fluor 488 goat anti mouse) in a ratio of 4:2:1, respectively, for20 minutes on ice. The complexes formed are diluted in 1% BSA-PBS to afinal HA concentration of 40, 20 or 10 μg/ml. Tissue sections are thenincubated with the HA-antibody complexes for 3 hours at RT. Sections arecounterstained with propidium iodide (Invitrogen; 1:100 in TBST), washedextensively and then viewed under a confocal microscope (Zeiss LSM510laser scanning confocal microscopy).

Precomplexed H5 HAs are also used along with other lectins such asJacalin (marker for non-ciliated mucinous cells such as goblet cells) toco-stain tissue sections to obtain additional information on whether HAstains ciliated and/or non-ciliated cells in the tissue epithelia.

Example 5 Testing H5 HA Polypeptide in an Animal Host

As described herein, the present invention encompasses the recognitionthat the use of animal hosts (e.g., ferrets) for the study oftransmission of virus may provide a reliable indicator of human virustransmission. Similarly, the present invention encompasses therecognition that the use of animal hosts (e.g., ferrets) treated withbinding agents in accordance with the invention (e.g., HA polypeptides)for the study of transmission of virus may provide a reliable indicatorof the efficacy of such binding agents in accordance with the inventionfor prevention or treatment of virus in a human host.

Virus Transmission Assay

A virus transmission assay is used in the presence or absence of bindingagents in accordance with the invention to determine viral transmissionin a suitable animal model. For example, animal hosts, e.g., ferrets,are housed in adjacent cages that prevent direct and indirect contactbetween animals. However, these housing conditions allow the spread ofinfluenza virus through the air. A first portion of the animals areinnoculated via methods known in the art (e.g., intranasally,intramuscularly, or any of the modes of administration described herein)with an effective amount of virus (“innoculated animals”). Naive animalscan then be introduced into cages adjacent to the innoculated animalsone, two, three or more days later.

Animals used in the study can be killed at any time one, two, three ormore days post-inoculation or transmission for analysis. Suitableanalysis for virus transmission studies can include, but is not limitedto determination of infectious virus titers (e.g., by nasal washes),observation of physical symptoms in the animals (e.g., lethargy,anorexia, rhinorrhea, sneezing, high fever, and/or death),immunohistochemical analysis of respiratory tissues, among others.

The virus transmission assay described above can also incorporate thetreatment of the animal host with a binding agent in accordance with theinvention described herein before, during or after inoculation ortransmission of virus. Analytic methods described herein are then usedto determine the efficacy of the binding agent(s) in blockingtransmission and/or infection of the animal host with the virus.

Serological Studies

Binding agents and/or vaccine compositions comprising binding agents areadministered intramuscularly in ferrets on day 0, followed by a boosterdose on day 21. Blood from each animal is recovered on days 0, 14, 21,and 35. The collected serum is examined in vitro for its ability toinhibit virus agglutination and neutralize virus infection.

Hemagglutination Inhibition (HAI) Assay

HAI titrations are performed in 96-well v-bottom plates (Corning). Seraare serially diluted 2-fold and added to 4 agglutinating doses ofinfluenza A virus in a total volume of 200 μl. Next, 25 μl of a 2%(vol/vol) erythrocyte solution is added. Sera, virus, and erythrocytesare gently mixed and the assay is read out after incubating for 30 min.Titers are recorded as the inverse of the highest antibody dilution thatinhibited 4 agglutinating doses of virus.

In Vitro Neutralization Assay

Serial dilutions of sera is mixed with viruses and incubated at roomtemperature for 30 min, and then incubated with MDCK cells for 1 hr at37° C. Cells are then washed twice with serum-free media, and then freshmedia with or without trypsin is added. Virus growth is scored bycytopathic effect. Data are expressed as the inverse dilution of highestdilution of sera that causes neutralization.

Virus Challenge Assay

Vaccinated ferrets are challenged intranasally with homologous andheterologous wild-type and mutant H5N1 strains. Nasal washes are takenfrom ferrets on days 1, 3, and 5 post-challenge. Virus is titrated inMDCK cells to determine virus shedding in the respiratory tract.

Example 5 A Two-Amino Acid Change in Recent Isolates of H5N1Hemagglutinin is Sufficient to Switch its Preference to Human Receptors

Introduction

Highly pathogenic H5N1 is a global concern that has initiated severallocalized outbreaks in humans since 2003 (Heumann et al., 2010, CellRes, 20:51; Guan et. al., 2009, Rev Sci Tech, 28:39; both of which arehereby incorporated by reference). Existing H5N1 strains are incapableof aerosol transmission but rather are primarily transmitted throughdirect contact with infected animals. However, the high morbidity andmortality rate associated with infection (˜60%) as well as the knownability of influenza subtypes (including H5N1) to acquire phenotypictraits through either mutation or gene reassortment, suggests that aH5N1 strain could acquire aerosol transmissibility (Yen et. al., 2009,Curr Top Microbiol Immunol, 333:3; incorporated herein by reference).Coupled with the potential for such a virus to cause severe infection,the fact that the human population has no pre-existing immunity to H5N1,suggests that a future epidemic or pandemic may occur should such astrain arise (Subbarao et. al., 2007, PLoS pathogens, 3:e40;incorporated herein by reference).

The use of reverse genetics systems has indicated that of the 11 geneproducts, acquisition of certain amino acid changes in hemagglutinin(HA) and the polymerase (PB2) are vital for human aerosol transmission(Hoeven et. al., 2009, Pro Natl Acad Sci USA, 106:3366; incorporatedherein by reference). Addressing the functional effect of geneticalterations in these proteins is thus especially important to identifythe potential for phenotypic alterations. In the case of PB2, a criticalalteration of lysine to glutamate at position 627 is key for acquiringaerosol transmissibility (Hoeven et. al., 2009, Pro Natl Acad Sci USA,106:3366; incorporated herein by reference). However, given thebiological role of HA, viz., binding to glycan receptors leading tofusion of the virion and infection, specific mutations that lead tohuman adaptation are thought to be subtype- and strain-specific (Stevenset. al., 2006, Nat Rev Microbiol, 4:857; Russell et. al., 2006,Glycoconj J, 23:85; both of which are hereby incorporated by reference).

Previous studies have identified that the receptor for HA are glycansterminated by particular glycan structures (e.g., “umbrella topology” or“cone topology” α2→3 or α2→6-linked sialic acid). Avian-adapted H5N1 HAspreferentially bind to glycan receptors terminated by cone topologyglycans, many of which have α2→3 linked sialic acid (avian receptors)(Stevens et al., 2006, J Mol Biol, 355:1143; Gambaryan et. al., 2006,Virology, 344:432; both of which are hereby incorporated by reference).HAs of human-adapted H1N1, H2N2, and H3N2 strains have a demonstratedswitch in binding preference from cone topology (e.g., many α2→3) toumbrella topology (e.g., many α2→6 sialylated glycans) (human receptors)and a characteristic high affinity binding to human receptors, whichhave been shown to correlate with the airborne transmissibility of thesehuman-adapted viruses (Pappas et. al., 2010, PLoS One, 5:e11158; Tumpeyet. al., 2007, Science, 315:655; both of which are hereby incorporatedby reference). Utilizing this framework, recent studies have identifiedsets of mutations which would lead to human adaptation of the currentlycirculating strains of H2N2, H7N7 and H9N2 (Viswanathan et. al., 2010,PLoS One, 5:e13768; Belser et. al., 2008, Proc Natl Acad Sci USA,105:7558; Sorrell et. al., 2009, Proc Natl Acad Sci USA, 106:7565; allof which are hereby incorporated by reference). These studiesdemonstrate that mutations required for conversion may differ based onthe subtype, and even the particular strain, studied. Previous studies(Maines et. al., 2011, Virology, 413:139; Stevens et. al., 2008, J MolBiol, 381:1382; Stevens et. al., 2006, Science, 312:404; all of whichare hereby incorporated by reference) that have mutated H5N1 strains toinclude either the hallmark changes for H2/H3 (Q226L and G228S or LS)and/or H1 (E190D, G225D or DD) mutations have shown that none of thesemutants quantitatively ‘switch’ to human receptor specificity andaffinity which is characteristic of human-adapted ‘pandemic’ strain HAs(FIG. 20). Attempts to introduce the hallmark LS residues on theA/Vietnam/1203/04 (Viet03_04) sequence have not yielded the switch. Thepresent inventors have established key structural features that areneeded to suitably accommodate the hallmark LS residues. The presentinventors have also determined which structural features are required tofacilitate a switch in receptor specificity as enable binding with highaffinity to human receptors.

Experimental Design

Influenza HA is a homotrimeric protein, wherein a monomer contains 552amino acids. Each monomer is composed of two disulphide-linked moieties,HA1 and HA2. HA1 comprises the glycan-receptor binding site (RBS),whereas HA2 is involved in the fusion of the viral and cellularmembranes. The RBS pocket involves HA positions 95, 131, 133, 136, 137,138, 145, 153, 155, 156, 158, 159, 183, 186, 187, 189, 190, 192, 193,194, 195, 196, 219, 222, 224, 225, 226, 227, 228 (H3 numbering used).

In order to determine structural features and correct H5 HA sequencesthat will accommodate the LS residues, the current inventors performed adetailed structural comparison of the RBS H2 and H5 HA, since H5 HA isclosest to H2 HA phylogenitically (FIG. 21). For this study, theprototypic pandemic H2 (A/Albany/6/58 or Alb6_58) HA with therepresentative H5N1 HA from an earlier human isolate (A/Vietnam/1203/04or Viet03_04) was chosen. Having made a comparative analysis, thecurrent study identified four distinct features which distinguish theRBS of H5 HA from that of H2 HA (FIG. 22). First, the composition of the130 loop of H2 HA is different from H5 HA, which includes a deletion atposition 130 (H3 numbering) in H5 HA. Second, there are differences inamino acid composition at the ‘top’ of the RBS or the ‘190-helix’ (suchas in positions 188, 192 and 193) that interact with sugar residuesbeyond the terminal Neu5Acα2-6Gal-motif of the human receptor. Third,there are differences in amino acid compositions at the ‘base’ of theRBS (such as in positions 137, 221, 226 and 228 which include LSchanges) that interact with the Neu5Acα2-6Gal-motif of the humanreceptor. Fourth, the glycosylation site at position 158 in H5 HA isabsent in H2 HA. Glycosylation at this site could potentially interferewith sugar residues beyond the terminal Neu5Acα2-6Gal-motif of the humanreceptor bound to the RBS (Stevens et. al., 2008, J Mol Biol, 381:1382;Wang et. al., 2010, Journal of Virology, 84:6570; both of which arehereby incorporated by reference). The inventive findings encompass thisdetailed comparison of RBS of H2 and H5 HA, which suggests particularamino acid differences that go beyond the more characteristic LS changespreviously observed. The present inventors sought to identifyappropriate H5 HA sequences that would facilitate matching thestructural features of H2 HA RBS in the context of the LS mutations.

Analysis of all the H5N1 sequences to-date (both avian and humanisolates) allowed the inventors to make three further observations: 1)H5 HAs from many of the recent avian and human isolates (after 2007)have already acquired the deletion in the 130-loop matching the firstfeature, 2) key amino acid changes were already observed in the‘190-helix’ matching feature 2, and 3) loss of glycosylation at 158position (feature 4) is also observed in many H5 HA sequences. In thecontext of the key structural features of HA RBS, the inventorsdetermined that the deletion in the 130 loop along with a loss ofglycosylation (features 1 and 4) concurrently in the same HA wascritical in the evolution of H5 HA. The present invention, however,encompasses the recognition that the loss of glycosylation isconcomitant with the deletion of 130-loop residue and not vice versa.The present invention encompasses the observation that specific currentH5 HA strains have diverged considerably from older human isolates (suchas Viet03_04), but have also acquired key structural features necessaryfor matching the pandemic H2 HA RBS.

Thus the present inventors assessed whether strains that have features 1and 4 would be the correct H5 HA sequence to suitably accommodate thehallmark LS residues. For the experiment strain A/chicken/Egypt/R2/2007(or ckEgy_07) was chosen as a representative H5 HA which naturallyacquired features 1 and 4 (FIG. 23). Introduction of just the LSmutation (ckEgy_07_LS) on this H5 HA sequence showed a switch and highaffinity binding to human receptors (and relatively poor affinity toavian receptors), thereby resembling the glycan binding characteristicsof human-adapted ‘pandemic’ HAs (FIGS. 24A, B and FIGS. 25A-D).Additional findings demonstrate binding of this mutant H5 HA to humanreceptors on the apical surface of the human tracheal epithelia (FIG.24C) that resembles the staining of this tissue by pandemic HAs(Viswanathan et. al., 2010, PLoS One, 5:e13768; Maines et. al., 2009,Science, 325:484; both of which are hereby incorporated by reference).

Earlier efforts by others to introduce the LS changes alone on olderhuman isolates such as Viet03_04 did not lead to a switch andunderscored the need to understand structural features of H5 HA RBS thatcan accommodate the LS mutations. Given that the (ckEgy_07_LS) alsonaturally acquired feature 4, the inventors assessed whether thisfeature alone i.e. loss of glycosylation (achieved through T160Amutation) together with LS switches the glycan receptor-bindingpreference of Viet03_04 (FIGS. 25A-D). Compared to Viet03_04 wild-typeHA, the dose-dependent direct glycan binding of this mutant strainshowed human receptor-binding but also retained its high affinity avianreceptor binding which is uncharacteristic of pandemic human-adapted HAs(FIGS. 25A-D). Introduction of the LS change alone on another strain(A/Egypt/2786-NAMRU3/06 or Egy_06), which naturally acquired feature 4,corroborates this observation. Finally, consistent with this structuralframework, mutations on a representative H5 HA(A/chicken/VietnamNCVD-093/08 or ckViet_08), which naturally acquiredfeature 2 (FIG. 22C) conferred human receptor binding affinity but alsoretained high affinity avian receptor binding (FIG. 26).

Discussion

The present inventors, for the first time, have defined certainstructural features that are needed to suitably accommodate the hallmarkLS residues. In addition, the inventors results provide the insight thatcertain current circulating H5 HAs only require two amino acid changes,the hallmark LS, to switch H5 HA preference to human receptors therebyleading to their human adaptation. Using the information from theinvention, the inventors rationally selected appropriate H5 HA sequences(albeit from a small representative pool of total H5N1 HAs) that wasamenable to switching receptor specificity by incorporating just the LSchanges. One characteristic property of these current H5 HAs is thatthey have naturally evolved to acquire features 1, 2 and 4 (FIGS.27A-B). Although, only 6% of the 2277 non-redundant H5N1 HA sequences inthe NCBI database have acquired features 1 & 4, however, it representsabout 45% of H5N1 strains isolated in 2009 and 2010. Furtherphylogenetic analyses of the H5N1 HAs reveal that the naturally acquiredfeatures 1 & 4 belong to clade 2.2.1. Thus far, occurrence of feature 1appears to be exclusive to clade 2.2.1, however the existence of feature4 is not restricted to 2.2.1. Critically, the clade 2.2.1 strains havealready diverged considerably from older human isolates (such asViet03_04) and are closer to human adaptation than these previousstrains. All of the reported human H5N1 isolates belonging to clade2.2.1 are from Egypt and Israel. Therefore, it is important to monitorthe evolution of the clade 2.2.1 strains.

Exemplary H5N1 Strains with a Postitively Charged Residue at Position129 (Feature 2)

ABJ96761/204-204 Avian China 2005 from clade 2.3.4; ABJ96763/204-204Avian China 2005 from clade 2.3.4; ABJ96764/204-204 Avian China 2006from clade 2.3.4; ACN39415/204-204 Avian China 2007 from clade 2.3.4;AC007037/204-204 Avian Viet Nam 2008 from clade 7; ADG28677/204-204Avian Egypt 2009 from clade 2.2.1; ADI58758/204-204 Avian Israel 2010from clade 2.2.1; ADM85869/204-204 Avian Egypt 2010 from clade 2.2.1;and ADG28684/204-204 Avian Egypt 2010 from clade 2.2.1.

Exemplary H5N1 Strains with a Deletion at 130 (and Loss of Glycosylationat 158) on the Same HA all Belonging to Clade 2.2.1(Feature 1). Includebut are not Limited to:

ABP96845 Human Egypt 2007 (SEQ ID NO. 122)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKRDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQXRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQXGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI >ABP96854 Human Egypt 2007(SEQ ID NO. 123)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ABM92273 Egypt 2007(SEQ ID NO. 124)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLNGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIAARSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15310 Human Egypt 2009(SEQ ID NO. 125)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRPSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPXDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIGNLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKMESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15312 Human Egypt 2009(SEQ ID NO. 126)MEKIVLLLAIVSIVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15314 Human Egypt 2009(SEQ ID NO. 127)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKITTRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15316 Human Egypt 2009(SEQ ID NO. 128)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLIPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15318 Human Egypt 2009(SEQ ID NO. 129)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15320 Human Egypt 2009(SEQ ID NO. 130)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGMSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15322 Human Egypt 2009(SEQ ID NO. 131)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLESRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15324 Human Egypt 2009(SEQ ID NO. 132)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAEEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNALERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15326 Human Egypt 2009(SEQ ID NO. 133)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLIPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15328 Human Egypt 2009(SEQ ID NO. 134)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPHYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15330 Human Egypt 2009(SEQ ID NO. 135)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPHYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15334 Human Egypt 2009(SEQ ID NO. 136)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKXVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMMAGLFLWMCSNGSLQCRICI >ACT15336 Human Egypt 2009(SEQ ID NO. 137)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15338 Human Egypt 2009(SEQ ID NO. 138)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNDATEQTRLYQNPTTYISVGTSTLNQRLIPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQEGRRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15340 Human Egypt 2009(SEQ ID NO. 139)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAEEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNALERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15342 Human Egypt 2009(SEQ ID NO. 140)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLILATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15343 Human Egypt 2009(SEQ ID NO. 141)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15345 Human Egypt 2009(SEQ ID NO. 142)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSSCPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFGSNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15349 Human Egypt 2009(SEQ ID NO. 143)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDATEQTRLYQNPTTYISVGTSTLNQRLIPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQEERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15353 Human Egypt 2009(SEQ ID NO. 144)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNSAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYSNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACT15357 Human Egypt 2009(SEQ ID NO. 145)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMERNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTQKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLIPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ABY79033 Avian Egypt 2007(SEQ ID NO. 146)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIKIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKKSTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEAKLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ABW37432 Avian Egypt 2007 (SEQ ID NO. 147)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTQISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKRRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ABW37433 Avian Egypt 2007 (SEQ ID NO. 148)MEKIVLLLAIVSLVESDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ABW37434 Avian Egypt 2007 (SEQ ID NO. 149)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGKRRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ABW37435 Avian Egypt 2007 (SEQ ID NO. 150)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVVSSLALAIMVAGLFLWMCSNGSLQCRICI >ABW37436 Avian Egypt 2007 (SEQ ID NO. 151)MEKIVLLLAIVSVVKSDQICIGYHANYSTEQVDTIMEKDVTVTHAQDILEKTHNGKLCNLEGMKPLILRDCSVAGWLLGNPMCDEFHNVPEWSYIVEKINPANDLCYPGNFDDYEELQHLFSRINHFEKIQIIPKNCWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGKRRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKEFGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACR56233 Avian Egypt 2008 (SEQ ID NO. 152)MEKIMLLLAIVSLVKGDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIVGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACR56248/1552 Avian Egypt 2008 (SEQ ID NO. 153)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYSNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLF >ACR56246 Avian Egypt 2008(SEQ ID NO. 154)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >AEA92628 Avian Egypt 2008 (SEQ ID NO. 155)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHETSGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDXAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYSNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACR56247 Avian Egypt 2008 (SEQ ID NO. 156)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKAHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLILWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACX31965 Avian Egypt 2009 (SEQ ID NO. 157)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYSNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADD21353/1565 Avian Egypt 2009 (SEQ ID NO. 158)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRI >ADD21367/1565 Avian Egypt 2009(SEQ ID NO. 159)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYSNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRI >ADD21378/1565 Avian Egypt 2009(SEQ ID NO. 160)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKSNPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRI >ACX31969 Avian Egypt 2009(SEQ ID NO. 161)MKKIVLLLAIVTLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI >ACX31970 Avian Egypt 2009 (SEQ ID NO. 162)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACX31978 Avian Egypt 2009 (SEQ ID NO. 163)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLIPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACX31989 Avian Egypt 2009 (SEQ ID NO. 164)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNSAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYSNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ACX31993 Avian Egypt 2009 (SEQ ID NO. 165)MEKIVLLLAIVSIVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNEQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADD21354/1565 Avian Egypt 2009 (SEQ ID NO. 166)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLIPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRI >ADD21355/1565 Avian Egypt 2009(SEQ ID NO. 167)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRI >ADD21361/1565 Avian Egypt 2009(SEQ ID NO. 168)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTXNQRLIPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRI >ADD21376/1559 Avian Egypt 2009(SEQ ID NO. 169)LLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPVNDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILRSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRI >ADD21379/1565 Avian Egypt 2009(SEQ ID NO. 170)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDDAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRI >ADD21380/1565 Avian Egypt 2009(SEQ ID NO. 171)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWITKKDNAYPTIRRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLIPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRI >ADD21371/1565 Avian Egypt 2009(SEQ ID NO. 172)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGISSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYSNCNTKCQTPIGAINSSMPFHNIHPITIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRI >ADD21377/1553 Avian Egypt 2009(SEQ ID NO. 173)LVKSDQICVGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLVTGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRI >ADY16730 Avian Egypt 2009(SEQ ID NO. 174)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDATEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADY16731 Avian Egypt 2009 (SEQ ID NO. 175)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDATEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVISSLALAIMVAGLFLWMCSNGSLQCRICI >ACX31975 Avian Egypt 2009 (SEQ ID NO. 176)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGIFLWMCSNGSLQCRICI >ACX31997/1553 Avian Egypt 2009 (SEQ ID NO. 177)KSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNYWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAEEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNALERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADD21359/1565 Avian Egypt 2009 (SEQ ID NO. 178)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLIPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVRLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRI >ADD21382/1565 Avian Egypt 2009(SEQ ID NO. 179)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDATEQTRLYQNPTTYISVGTSTLNQRLIPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQEERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRI >ADB77952/1561 Avian Egypt 2009(SEQ ID NO. 180)LLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNGPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADB85109 Avian Egypt 2009 (SEQ ID NO. 181)MEKMVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNGPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85844 Avian Egypt 2009 (SEQ ID NO. 182)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNSAYPTIKKSYNNTNQEDLLVLWGIHHPNDAEEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNALERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI >ADM85845 Avian Egypt 2009 (SEQ ID NO. 183)MEKIVLLLAIVSIVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLNGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85847 Avian Egypt 2010 (SEQ ID NO. 184)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDATEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFDSNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85852 Avian Egypt 2010 (SEQ ID NO. 185)MEKIVLLLAIVSIVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRPKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85854 Avian Egypt 2010 (SEQ ID NO. 186)MEKIVLLLAIVSIVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIANRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85855 Avian Egypt 2010 (SEQ ID NO. 187)MEKIVLLLAIVSIVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85856/1557 Avian Egypt 2010 (SEQ ID NO. 188)VSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDATEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGDRRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85861 Avian Egypt 2010 (SEQ ID NO. 189)MEKIVLLLAIFSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85862 Avian Egypt 2010 (SEQ ID NO. 190)MEKIVLLLAIVSIVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPINDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85863 Avian Egypt 2010 (SEQ ID NO. 191)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDATEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGDRRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85868 Avian Egypt 2010 (SEQ ID NO. 192)MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHGASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKKSYNNTNQEDLLILWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85870 Avian Egypt 2010 (SEQ ID NO. 193)MEKIVLLLAIVSIVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKKSYNNTNQEDLLVLWGIHHPNDAVEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLVLAIIVAGLFLWMCSNGSLQCRICI >ADM85871 Avian Egypt 2010 (SEQ ID NO. 194)MEKIVLLLAIFSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYLTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85873/1558 Avian Egypt 2010 (SEQ ID NO. 195)IVSIVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKDSWSDHEASGVSSACPYQGRSSFFRNVVWLTKRNNAYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85874 Avian Egypt 2010 (SEQ ID NO. 196)MEKIVLLLAIVSIVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKDSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKKNYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85875 Avian Egypt 2010 (SEQ ID NO. 197)MEKIVLLLAIVSIVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPSNDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKKSYNNTNQEDLLVLWGIHHPNDEAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIVPENAYKIVKKGDSTIMKSELEYGSCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERKRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85876 Avian Egypt 2010 (SEQ ID NO. 198)MEKIVLLLAIISIVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCSLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85878 Avian Egypt 2010 (SEQ ID NO. 199)MEKIVLLLAIVSIVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85884 Avian Egypt 2010 (SEQ ID NO. 200)MEKIVLLLAIVSIVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIISKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVVSSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85881 Avian Egypt 2010 (SEQ ID NO. 201)MEKIVLLLAIFSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRNCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADM85883 Avian Egypt 2010 (SEQ ID NO. 202)MEKIVLLLAIVSIVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLNGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLFLWMCSNGSLQCRICI >ADQ53454 Avian Israel 2010 (SEQ ID NO. 203)MEKIVLLLAIVSIVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNNAYPTIKKSYNNTNQEDLLVLWGIHHPNDEAEQTRLYQNSTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLKDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVTSSLALAIMVAGLFLWMCSNGSLQCRICI >AEN68621 Avian Israel 2011 (SEQ ID NO. 204)MEKIVLLLAIVSIVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKNSWSDHEASGVSSACPYQGRSSFFRNVVWLTKKNDAYPTIKKSYNNTNQEDLLVIWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYSNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGEKRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLVLAIMVAGLFLWMCSNGSLQCRICI

Materials and Methods

Cloning, Baculovirus Synthesis, Expression and Purification of HA

H5 WT and mutant HA sequences were codon-optimized for insect cellexpression and synthesized at DNA2.0 (Menlo Park, Calif.). Thesynthesized genes were then sub-cloned into pAcGP67A plasmid andbaculoviruses were created using Baculogold system (BD Biosciences, SanJose, Calif.) according to manufacturer's instructions. The recombinantbaculoviruses were then used to infect suspension cultures of Sf9 cellscultured in BD Baculogold Max-XP SFM (BD Biosciences, San Jose, Calif.).The infection was monitored and the conditioned media was harvested 3-4days post-infection. The soluble HA from the harvested conditioned mediawas purified using Nickel affinity chromatography (HisTrap HP columns,GE Healthcare, Piscataway, N.J.). Eluting fractions containing HA werepooled, concentrated and buffer exchanged into 1× PBS pH 8.0 (Gibco)using 100K MWCO spin columns (Millipore, Billerica, Mass.). The purifiedprotein was quantified using BCA method (Pierce).

Binding of Recombinant HA to Human Tracheal Tissue Sections

Paraffinized human tracheal (US Biological) tissue sections weredeparaffinized, rehydrated and incubated with 1% BSA in PBS for 30minutes to prevent non-specific binding. HA was pre-complexed withprimary antibody (mouse anti 6× His tag (SEQ ID NO: 207), Abcam) andsecondary antibody (Alexa fluor 488 goat anti mouse, Invitrogen) in amolar ratio of 4:2:1, respectively, for 20 minutes on ice. The tissuebinding was performed over different HA concentrations by diluting thepre-complexed HA in 1% BSA-PBS. Tissue sections were then incubated withthe HA-antibody complexes for 3 hours at RT. The tissue sections werecounterstained by propidium iodide (Invitrogen; 1:100 in TBST). Thetissue sections were mounted and then imaged using a confocal microscope(Zeiss LSM510 laser scanning confocal microscopy).

Dose Dependent Direct Binding of WT and Mutant HA

To investigate the multivalent HA-glycan interactions a streptavidinplate array comprising representative biotinylated α2→3 and α2→6sialylated glycans was used as described previously. 3′SLN, 3′SLN-LN,3′SLN-LN-LN are representative avian receptors. 6′SLN and 6′SLN-LN arerepresentative human receptors. LN corresponds to lactosamine(Galβ1-4GlcNAc) and 3′SLN and 6′SLN respectively correspond toNeu5Acα2-3 and Neu5Acα2-6 linked to LN (FIG. 28). The biotinylatedglycans were obtained from the Consortium of Functional Glycomicsthrough their resource request program. Streptavidin-coated High BindingCapacity 384-well plates (Pierce) were loaded to the full capacity ofeach well by incubating the well with 50 μl of 2.4 μM of biotinylatedglycans overnight at 4° C. Excess glycans were removed through extensivewashing with PBS. The trimeric HA unit comprises of three HA monomers(and hence three RBS, one for each monomer). The spatial arrangement ofthe biotinylated glycans in the wells of the streptavidin plate arrayfavors binding to only one of the three HA monomers in the trimeric HAunit. Therefore in order to specifically enhance the multivalency in theHA-glycan interactions, the recombinant HA proteins were pre-complexedwith the primary and secondary antibodies in the molar ratio of 4:2:1(HA:primary:secondary). The identical arrangement of 4 trimeric HA unitsin the precomplex for all the HAs permits comparison between theirglycan binding affinities. A stock solution containing appropriateamounts of Histidine tagged HA protein, primary antibody (Mouse anti 6×His tag (SEQ ID NO: 207) IgG from Abcam) and secondary antibody (HRPconjugated goat anti Mouse IgG from Santacruz Biotechnology) in theratio 4:2:1 and incubated on ice for 20 min. Appropriate amounts ofprecomplexed stock HA were diluted to 250 μl with 1% BSA in PBS. 50 μlof this precomplexed HA was added to each of the glycan-coated wells andincubated at room temperature for 3 hrs followed by the wash steps withPBS and PBST (1× PBS+0.05% Tween-20). The binding signal was determinedbased on HRP activity using Amplex Red Peroxidase Assay kit (Invitrogen,CA) according to the manufacturer's instructions.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to embodiments ofthe invention described herein. The scope of the present invention isnot intended to be limited to the above Description, but rather is asset forth in the following claims:

1.-20. (canceled)
 21. A method of inhibiting binding of a H5 influenzavirus to a hemagglutinin receptor having an umbrella topology, in asubject, or minimizing the risk of infection of a subject by aninfluenza virus which binds a hemagglutinin receptor having an umbrellatopology, or treating a subject, the method comprising: identifying asubject susceptible to or suffering from infection by an influenzavirus, selecting a binding agent that competes a glycan HA polypeptideinteraction between an umbrella topology glycan and an HA polypeptide;and administering an effective amount of the binding agent so thatbinding by the virus to hemagglutinin receptors having umbrella topologyglycans is reduced, the risk is minimized, or the patient is treated.22. The method of claim 21, wherein the binding agent comprises an H5 HApolypeptide sequence from a reference H5 HA polypeptide sequence at oneor more of residues selected from the group consisting of residues 130,131, 132, 133, 135, 137, 155, 188, 192, 193, 221, 226, 227, 228, andcombinations thereof.
 23. The method of claim 21, wherein the step ofselecting includes selecting a binding agent on the basis of the bindingagent being able to bind a hemagglutinin receptor having umbrellatopology glycans.
 24. The method of claim 21, wherein the binding agentis an LSBA.
 25. The method of claim 21, wherein the binding agent is aUTBA.
 26. The method of claim 21, wherein the binding agent is a UTSBA.27. The method of claim 26, wherein the UTSBA is administered to thesubject prior to exposure to the virus.
 28. The method of claim 26,wherein the UTSBA is administered to the subject after exposure to thevirus.
 29. The method of claim 21, wherein amount administered issufficient to saturate the subject's HA receptors containing umbrellatopology glycans.
 30. The method of claim 26, wherein the UTSBA isadministered by inhalation.
 31. The method of claim 26, wherein theUTSBA is selected from the group consisting of antibodies, lectins,aptamers, and non-HA polypeptides.
 32. The method of claim 26, whereinsaid UTSBA is administered in combination with administration of asecond therapeutic.
 33. The method of claim 32, wherein the secondtherapeutic is an anti-viral agent.